Glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthases

ABSTRACT

Genes encoding Class II EPSPS enzymes are disclosed. The genes are useful in producing transformed bacteria and plants which are tolerant to glyphosate herbicide. Class II EPSPS genes share little homology with known, Class I EPSPS genes, and do not hybridize to probes from Class I EPSPS&#39;s. The Class II EPSPS enzymes are characterized by being more kinetically efficient than Class I EPSPS&#39;s in the presence of glyphosate. Plants transformed with Class II EPSPS genes are also disclosed as well as a method for selectively controlling weeds in a planted transgenic crop field.

This is a continuation-in-part of a U.S. patent application Ser. No.07/749,611, filed Aug. 28, 1991 now abandoned, which is acontinuation-in-part of U.S. patent application Ser. No. 07/576,537,filed Aug. 31, 1990, now abandoned.

BACKGROUND OF THE INVENTION

This invention relates in general to plant molecular biology and, moreparticularly, to a new class of glyphosate-tolerant5-enolpyruvylshikimate-3-phosphate synthases.

Recent advances in genetic engineering have provided the requisite toolsto transform plants to contain foreign genes. It is now possible toproduce plants which have unique characteristics of agronomicimportance. Certainly, one such advantageous trait is more costeffective, environmentally compatible weed control via herbicidetolerance. Herbicide-tolerant plants may reduce the need for tillage tocontrol weeds thereby effectively reducing soil erosion.

One herbicide which is the subject of much investigation in this regardis N-phosphonomethylglycine commonly referred to as glyphosate.Glyphosate inhibits the shikimic acid pathway which leads to thebiosynthesis of aromatic compounds including amino acids, plant hormonesand vitamins. Specifically, glyphosate curbs the conversion ofphosphoenolpyruvic acid (PEP) and 3-phosphoshikimic acid to5-enolpyruvyl-3-phosphoshikimic acid by inhibiting the enzyme5-enolpyruvylshikimate-3-phosphate synthase (hereinafter referred to asEPSP synthase or EPSPS). For purposes of the present invention, the term“glyphosate” should be considered to include any herbicidally effectiveform of N-phosphonomethylglycine (including any salt thereof) and otherforms which result in the production of the glyphosate anion in plants.

It has been shown that glyphosate-tolerant plants can be produced byinserting into the genome of the plant the capacity to produce a higherlevel of EPSP synthase in the chloroplast of the cell (Shah et al.,1986) which enzyme is preferably glyphosate-tolerant (Kishore et al.1988). Variants of the wild-type EPSPS enzyme have been isolated whichare glyphosate-tolerant as a result of alterations in the EPSPS aminoacid coding sequence (Kishore and Shah, 1988; Schulz et al., 1984; Sostet al., 1984; Kishore et al., 1986). These variants typically have ahigher K_(i) for glyphosate than the wild-type EPSPS enzyme whichconfers the glyphosate-tolerant phenotype, but these variants are alsocharacterized by a high K_(m) for PEP which makes the enzyme kineticallyless efficient (Kishore and Shah, 1988; Sost et al., 1984; Schulz etal., 1984; Kishore et al., 1986; Sost and Amrhein, 1990). For example,the apparent K_(m) for PEP and the apparent K_(i) for glyphosate for thenative EPSPS from E. coli are 10 μM and 0.5 μM while for aglyphosate-tolerant isolate having a single amino acid substitution ofan alanine for the glycine at position 96 these values are 220 μM and4.0 mM, respectively. A number of glyphosate-tolerant plant variantEPSPS genes have been constructed by mutagenesis. Again, theglyphosate-tolerant EPSPS was impaired due to an increase in the K_(m)for PEP and a slight reduction of the V_(max) of the native plant enzyme(Kishore and Shah, 1988) thereby lowering the catalytic efficiency(V_(max)/K_(m)) of the enzyme. Since the kinetic constants of thevariant enzymes are impaired with respect to PEP, it has been proposedthat high levels of overproduction of the variant enzyme, 40-80 fold,would be required to maintain normal catalytic activity in plants in thepresence of glyphosate (Kishore et al., 1988).

While such variant EPSP synthases have proved useful in obtainingtransgenic plants tolerant to glyphospate, it would be increasinglybeneficial to obtain an EPSP synthase that is highly glyphosate-tolerantwhile still kinetically efficient such that the amount of theglyphosate-tolerant EPSPS needed to be produced to maintain normalcatalytic activity in the plant is reduced or that improved tolerance beobtained with the same expression level.

Previous studies have shown that EPSPS enzymes from different sourcesvary widely with respect to their degree of sensitivity to inhibition byglyphosate. A study of plant and bacterial EPSPS enzyme activity as afunction of glyphosate concentration showed that there was a very widerange in the degree of sensitivity to glyphosate. The degree ofsensitivity showed no correlation with any genus or species tested(Schulz et al., 1985). Insensitivity to glyphosate inhibition of theactivity of the EPSPS from the Pseudomonas sp. PG2982 has also beenreported but with no details of the studies (Fitzgibbon, 1988). Ingeneral, while such natural tolerance has been reported, there is noreport suggesting the kinetic superiority of the naturally occurringbacterial phosphosate-tolerant EPSPS enzymes over those of mutated EPSPSenzymes nor have any of the genes been characterized. Similarly, thereare no reports on the expression of naturally glyphosate-tolerant EPSPSenzymes in plants to confer glyphosate tolerance.

For purposes of the present invention the term “mature EPSP synthase”relates to the EPSPS polypeptide without the N-terminal chloroplasttransit peptide. It is now known that the precursor form of the EPSPsynthase in plants (with the transit peptide) is expressed and upondelivery to the chloroplast, the transit peptide is cleaved yielding themature EPSP synthase. All numbering of amino acid positions are givenwith respect to the mature EPSP synthase (without chloroplast transitpeptide leader) to facilitate comparison of EPSPS sequences from sourceswhich have chloroplast transit peptides (i.e., plants and fungi) tosources which do not utilize a chloroplast targeting signal (i.e.,bacteria).

In the amino acid sequences which follow, the standard single letter orthree letter nomenclature are used. All peptide structures representedin the following description are shown in conventional format in whichthe amino group at the N-terminus appears to the left and the carboxylgroup at the C-terminus at the right. Likewise, amino acid nomenclaturefor the naturally occurring amino acids found in protein is as follows:alanine (Ala;A), asparagine (Asn;N), aspartic acid (Asp;D), arginine(Arg;R), cysteine (Cys;C), glutamic acid (Glu;E), glutamine (Gln;Q),glycine (Gly;G), histidine (His;H), isoleucine (Ile;I), leucine (Leu;L),lysine (Lys;k), methionine (Met;M), phenylalanine (Phe;F), proline(Pro;P), serine (Ser;S), threonine (Thr;T), tryptophan (Trp;W), tyrosine(Tyr;Y), and valine (Val;V). An “X” is used when the amino acid residueis unknown and parentheses designate that an unambiguous assignment isnot possible and the amino acid designation within the parentheses isthe most probable estimate based on known information.

The term “nonpolar” amino acids include alanine, valine, leucine,isoleucine, proline, phenylalanine, tryptophan, and methionine. The term“uncharged polar” amino acids include glycine, serine, threonine,cysteine, tyrosine, asparagine and glutamine. The term “charged polar”amino acids includes the “acidic” and “basic” amino acids. The term“acidic” amino acids includes aspartic acid and glutamic acid. The term“basic” amino acid includes lysine, arginine and histidine. The term“polar” amino acids includes both “charged polar” and “uncharged polar”amino acids.

Deoxyribonucleic acid (DNA) is a polymer comprising four mononucleotideunits, dAMP (2′-Deoxyadenosine-5-monophosphate), dGMP(2′-Deoxyguanosine-5-monophosphate), dCMP(2′-Deoxycytosine-5-monophosphate) and dTMP(2′-Deoxythymosine-5-monophosphate) linked in various sequences by3′,5′-phosphodiester bridges. The structural DNA consists of multiplenucleotide triplets called “codons” which code for the amino acids. Thecodons correspond to the various amino acids as follows: Arg (CGA, CGC,CGG, CGT, AGA, AGG); Leu (CTA, CTC, CTG, CTT, TTA, TTG); Ser (TCA, TCC,TCG, TCT, AGC, AGT); Thr (ACA, ACC, ACG, ACT); Pro (CCA, CCC, CCG, CCT);Ala (GCA, GCC, GCG, GCT); Gly (GGA, GGC, GGG, GGT); Ile (ATA, ATC, ATT);Val (GTA, GTC, GTG, GTT); Lys (AAA, AAG); Asn (AAC, AAT); Gla (CAA,CAG); His (CAC, CAT); Glu (GAA, GAG); Asp (GAC, GAT); Tyr (TAC, TAT);Cys (TGC, TGT); Phe (TTC, TTT); Met (ATG); and Trp (UGG). Moreover, dueto the redundancy of the genetic code (i.e., more than one codon for allbut two amino acids), there are many possible DNA sequences which maycode for a particular amino acid sequence.

SUMMARY OF THE INVENTION

DNA molecules comprising DNA encoding kinetically efficient,glyphosate-tolerant EPSP synthases are disclosed. The EPSP synthases ofthe present invention reduce the amount of overproduction of the EPSPSenzyme in a transgenic plant necessary for the enzyme to maintaincatalytic activity while still conferring glyphosate tolerance. The EPSPsynthases described herein represent a new class of EPSPS enzymes,referred to hereinafter as Class II EPSPS enzymes. Class II EPSPSenzymes of the present invention usually share only between about 47%and 55% amino acid similarity or between about 22% and 30% amino acididentity to other known bacterial or plant EPSPS enzymes and exhibittolerance to glyphosate while maintaining suitable K_(m) (PEP) ranges.Suitable ranges of K_(m) (PEP) for EPSPS for enzymes of the presentinvention are between 1-150 μM, with a more preferred range of between1-35 μM, and a most preferred range between 2-25 μM. These kineticconstants are determined under the assay conditions specifiedhereinafter. An EPSPS of the present invention preferably has a K_(i)for glyphosate range of between 15-10000 μM. The K_(i)/K_(m) ratioshould be between about 2-500, and more preferably between 25-500. TheV_(max) of the purified enzyme should preferably be in the range of2-100 units/mg (μmoles/minute.mg at 25° C.) and the K_(m) forshikimate-3-phosphate should preferably be in the range of 0.1 to 50 μM.

Genes coding for Class II EPSPS enzymes have been isolated from five (5)different bacteria:Agrobacterium tumefaciens sp. strain CP4,Achromobacter sp. strain LBAA, Pseudomonas sp. strain PG2982, Bacillussubtilis, and Staphylococcus aureus. The LBAA and PG2982 Class II EPSPSgenes have been determined to be identical and the proteins encoded bythese two genes are very similar to the CP4 protein and shareapproximately 84% amino acid identity with it. Class II EPSPS enzymesoften may be distinguished from Class I EPSPS's by their inability toreact with polyclonal antibodies prepared from Class I EPSPS enzymesunder conditions where other Class I EPSPS enzymes would readily reactwith the Class I antibodies as well as the presence of certain uniqueregions of amino acid homology which are conserved in Class II EPSPsynthases as discussed hereinafter.

Other Class II EPSPS enzymes can be readily isolated and identified byutilizing a nucleic acid probe from one of the Class II EPSPS genesdisclosed herein using standard hybridization techniques. Such a probefrom the CP4 strain has been prepared and utilized to isolate the ClassII EPSPS genes from strains LBAA and PG2982. These genes may alsooptionally be adapted for enhanced expression in plants by knownmethodology. Such a probe has also been used to identify homologousgenes is bacteria isolated de novo from soil.

The Class II EPSPS enzymes are preferably fused to a chloroplast transitpeptide (CTP) to target the protein to the chloroplasts of the plantinto which it may be introduced. Chimeric genes encoding this CTP-ClassII EPSPS fusion protein may be prepared with an appropriate promoter and3′ polyadenylation site for introduction into a desired plant bystandard methods.

To obtain the maximal tolerance to glyphosate herbicide it is preferableto transform the desired plant with a plant-expressible Class II EPSPSgene in conjunction with another plant-expressible gene which expressesa protein capable of degrading glyphosate such as a plant-expressiblegene encoding a glyphosate oxidoreductase enzyme as described in PCTApplication No. WO 92/00377, the disclosure of which is herebyincorporated by reference.

Therefore, in one aspect, the present invention provides a new class ofEPSP synthases that exhibit a low K_(m) for phosphoenolpyruvate (PEP), ahigh V_(max)/K_(m) ratio, and a high K_(i) for glyphosate such that whenintroduced into a plant, the plant is made glyphosate-tolerant such thatthe catalytic activity of the enzyme and plant metabolism are maintainedin a substantially normal state. For purposes of this discussion, ahighly efficient EPSPS refers to its efficiency in the presence ofglyphosate.

More particularly, the present invention provides EPSPS enzymes having aK_(m) for phosphoenolpyruvate (PEP) between 1-150 μM and aK_(i)(glyphosate)/K_(m) (PEP) ratio between 3-500, said enzymes havingthe sequence domains:

-   -   -R-X₁-H-X₂-E-(SEQ ID NO:37), in which        -   X₁ is an uncharged polar or acidic amino acid,        -   X₂ is serine or threonine; and    -   -G-D-K-X₃-(SEQ ID NO:38), in which        -   X₃ is serine or threonine; and    -   -S-A-Q-X₄-K-(SEQ ID NO:39), in which        -   X₄ is any amino acid; and    -   -N-X₅-T-R-(SEQ ID NO:40), in which        -   X₅ is any amino acid.

Exemplary Class II EPSPS enzyme sequences are disclosed from sevensources: Agrobacterium sp. strain designated CP4, Achromobacter sp.strain LBAA, Pseudomonas sp. strain PG2982, Bacillus subtilis 1A2,Staphylococcus aureus (ATCC 35556), Synechocystis sp. PCC6803 andDichelobacter nodosus.

In another aspect of the present invention, a double-stranded DNAmolecule comprising DNA encoding a Class II EPSPS enzyme is disclosed.Exemplary Class II EPSPS enzyme DNA sequences are disclosed from sevensources: Agrobacterium sp. strain designated CP4, Achromobacter sp.strain LBAA, Pseudomonas sp. strain PG2982, Bacillus subtilis 1A2,Staphylococcus aureus (ATCC 35556), Synechocystis sp. PCC6803 andDichelobacter nodosus.

In a further aspect of the present invention, nucleic acid probes fromEPSPS Class II genes are presented that are suitable for use inscreening for Class II EPSPS genes in other sources by assaying for theability of a DNA sequence from the other source to hybridize to theprobe.

In yet another aspect of the present invention, a recombinant,double-stranded DNA molecule comprising in sequence:

-   -   a) a promoter which functions in plant cells to cause the        production of an RNA sequence;    -   b) a structural DNA sequence that causes the production of an        RNA sequence which encodes a Class II EPSPS enzyme having the        sequence domains;        -   -R-X₁-H-X₂-E-(SEQ ID NO:37), in which            -   X₁ is an uncharged polar or acidic amino acid,            -   X₂ is serine or threonine; and        -   -G-D-K-X₃-(SEQ ID NO:38), in which            -   X₃ is serine or threonine; and        -   -S-A-Q-X₄-K-(SEQ ID NO:39), in which            -   X₄ is any amino acid; and        -   -N-X₅-T-R-(SEQ ID NO:40), in which            -   X₅ is any amino acid; and    -   c) a 3′ nontranslated region which functions in plant cells to        cause the addition of a stretch of polyadenyl nucleotides to the        3′ end of the RNA sequence        where the promoter is heterologous with respect to the        structural DNA sequence and adapted to cause sufficient        expression of the EPSP synthase polypeptide to enhance the        glyphosate tolerance of a plant cell transformed with said DNA        molecule.

In still yet another aspect of the present invention, transgenic plantsand transformed plant cells are disclosed that are madeglyphosate-tolerant by the introduction of the above-describedplant-expressible Class II EPSPS DNA molecule into the plant's genome.

In still another aspect of the present invention, a method forselectively controlling weeds in a crop field is presented by plantingcrop seeds or crop plants transformed with a plant-expressible Class IIEPSPS DNA molecule to confer glyphosate tolerance to the plants whichallows for glyphosate containing herbicides to be applied to the crop toselectively kill the glyphosate sensitive weeds, but not the crops.

Other and further objects, advantages and aspects of the invention willbecome apparent from the accompanying drawing figures and thedescription of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B, show the DNA sequence (SEQ ID NO:1) for the full-lengthpromoter of figwort mosaic virus (FMV35S).

FIG. 2 shows the cosmid cloning vector pMON17020.

FIG. 3A, 3B, 3C, 3D and 3E show the structural DNA sequence (SEQ IDNO:2) for the Class II EPSPS gene from bacterial isolate Agrobacteriumsp. strain CP4 and the deduced amino acid sequence (SEQ ID NO:3).

FIG. 4A, 4B, 4C, 4D and 4E show the structural DNA sequence (SEQ IDNO:4) for the Class II EPSPS gene from the bacterial isolateAchromobacter sp. strain LBAA and the deduced amino acid sequence (SEQID NO:5).

FIG. 5A, 5B, 5C, 5D and 5E show the structural DNA sequence (SEQ IDNO:6) for the Class II EPSPS gene from the bacterial isolate Pseudomonassp. strain PG2982 and the deduced amino acid sequence (SEQ ID NO:7).

FIG. 6A and 6B show the Bestfit comparison of the CP4 EPSPS amino acidsequence (SEQ ID NO:3) with that for the E. coli EPSPS (SEQ ID NO:8).

FIG. 7A and 7B show the Bestfit comparison of the CP4 EPSPS amino acidsequence (SEQ ID NO:3) with that for the LBAA EPSPS (SEQ ID NO:5).

FIG. 8A and 8B show the structural DNA sequence (SEQ ID NO:9) for thesynthetic CP4 Class II EPSPS gene.

FIG. 9 shows the DNA sequence (SEQ ID NO:10) of the chloroplast transitpeptide (CTP) and encoded amino acid sequence (SEQ ID NO:11) derivedfrom the Arabidopsis thaliana EPSPS CTP and containing a SphIrestriction site at the chloroplast processing site, hereinafterreferred to as CTP2.

FIG. 10A and 10B show the DNA sequence (SEQ ID NO:12) of the chloroplasttransit peptide and encoded amino acid sequence (SEQ ID NO:13) derivedfrom the Arabidopsis thaliana EPSPS gene and containing an EcoRIrestriction site within the mature region of the EPSPS, hereinafterreferred to as CTP3.

FIG. 11 shows the DNA sequence (SEQ ID NO:14) of the chloroplast transitpeptide and encoded amino acid sequence (SEQ ID NO:15) derived from thePetunia hybrida EPSPS CTP and containing a SphI restriction site at thechloroplast processing site and in which the amino acids at theprocessing site are changed to -Cys-Met-, hereinafter referred to asCTP4.

FIG. 12A and 12B show the DNA sequence (SEQ ID NO:16) of the chloroplasttransit peptide and encoded amino acid sequence (SEQ ID NO:17) derivedfrom the Petunia hybrida EPSPS gene with the naturally occurring EcoRIsite in the mature region of the EPSPS gene, hereinafter referred to asCTP5.

FIG. 13 shows a plasmid map of CP4 plant transformation/expressionvector pMON17110.

FIG. 14 shows a plasmid map of CP4 synthetic EPSPS gene planttransformation/expression vector pMON17131.

FIG. 15 shows a plasmid map of CP4 EPSPS free DNA plant transformationexpression vector pMON13640.

FIG. 16 shows a plasmid map of CP4 plant transformation/direct selectionvector pMON17227.

FIG. 17 shows a plasmid map of CP4 plant transformation/expressionvector pMON19653.

FIG. 18A, 18B, 18C and 18D show the structural DNA sequence (SEQ IDNO:41) for the Class II EPSPS gene from the bacterial isolate Bacillussubtilis and the deduced amino acid sequence (SEQ ID NO:42).

FIG. 19A, 19B, 19C and 19D show the structural DNA sequence (SEQ IDNO:43) for the Class II EPSPS gene from the bacterial isolateStaphylococcus aureus and the deduced amino acid sequence (SEQ IDNO:44).

FIG. 20A, 20B, 20C, 20D, 20E, 20F, 20G, 20H, 20I, 20J and 20K show theBestfit comparison of the representative Class II EPSPS amino acidsequences Pseudomonas sp. stain PG2982 (SEQ ID NO:7), Achromobacter sp.strain LBAA (SEQ ID NO:5), Agrobacterium sp. strain designated CP4 (SEQID NO:3), Bacillus subtilis (SEQ ID NO:42), and Staphylococcus aureus(SEQ ID NO:44) with that for representative Class I EPSPS amino acidsequences [Sacchromyces cerevisiae (SEQ ID NO:49), Aspergillus nidulans(SEQ ID NO:50), Brassica napus (SEQ ID NO:51), Arabidopsis thaliana (SEQID NO:52), Nicotina tobacum (SEQ ID NO:53), L. esculentum (SEQ IDNO:54), Petunia hybrida (SEQ ID NO:55), Zea mays (SEQ ID NO:56),Solmenella gallinarum (SEQ ID NO:57), Solmenella typhimurium (SEQ IDNO:58), Solmenella typhi (SEQ ID NO:65), E. coli (SEQ ID NO:8), K.pneumoniae (SEQ ID NO:59), Y. enterocolitica (SEQ ID NO:60), H.influenzae (SEQ ID NO:61), P. multocida (SEQ ID NO:62), Aeromonassalmonicida (SEQ ID NO:63), Bacillus pertussis (SEQ ID NO:64)] andillustrates the conserved regions among Class II EPSPS sequences whichare unique to Class II EPSPS sequences. To aid in a comparison of theEPSPS sequences, only mature EPSPS sequences were compared. That is, thesequence corresponding to the chloroplast transit peptide, if present ina subject EPSPS, was removed prior to making the sequence alignment.

FIG. 21A, 21B, 21C, 21D and 21E show the structural DNA sequence (SEQ IDNO:66) for the Class II EPSPS gene from the bacterial isolateSynechocystis sp. PCC6803 and the deduced amino acid sequence (SEQ IDNO:67).

FIG. 22A, 22B, 22C, 22D and 22E show the structural DNA sequence (SEQ IDNO:68) for the Class II EPSPS gene from the bacterial isolateDichelobacter nodosus and the deduced amino acid sequence (SEQ IDNO:69).

FIG. 23A, 23B, 23C and 23D show the Bestfit comparison of therepresentative Class II EPSPS amino acid sequences Pseudomonas sp.strain PG2982 (SEQ ID NO:7), Achromobacter sp. strain LBAA (SEQ IDNO:5), Agrobacterium sp. strain designated CP4 (SEQ ID NO:3),Synechocystis sp. PCC6803 (SEQ ID NO:67), Bacillus subtilis (SEQ IDNO:42), Dichelobacter nodosus (SEQ ID NO:69) and Staphylococcus aureus(SEQ ID NO:44).

FIG. 24 a plasmid map of canola plant transformation/expression vectorpMON17209.

FIG. 25 a plasmid map of canola plant transformation/expression vectorpMON17237.

STATEMENT OF THE INVENTION

The expression of a plant gene which exists in double-stranded DNA forminvolves synthesis of messenger RNA (mRNA) from one strand of the DNA byRNA polymerase enzyme, and the subsequent processing of the mRNA primarytranscript inside the nucleus. This processing involves a 3′non-translated region which adds polyadenylate nucleotides to the 3′ endof the RNA.

Transcription of DNA into mRNA is regulated by a region of DNA usuallyreferred to as the “promoter.” The promoter region contains a sequenceof bases that signals RNA polymerase to associate with the DNA, and toinitiate the transcription into mRNA using one of the DNA strands as atemplate to make a corresponding complementary strand of RNA. A numberof promoters which are active in plant cells have been described in theliterature. These include the nopaline synthase (NOS) and octopinesynthase (OCS) promoters (which are carried on tumor-inducing plasmidsof Agrobacterium tumefaciens), the cauliflower mosaic virus (CaMV) 19Sand 35S promoters, the light-inducible promoter from the small subunitof ribulose bis-phosphate carboxylase (ssRUBISCO, a very abundant plantpolypeptide) and the full-length transcript promoter from the figwortmosaic virus (FMV35S), promoters from the maize ubiquitin and rice actingenes. All of these promoters have been used to create various types ofDNA constructs which have been expressed in plants; see, e.g., PCTpublication WO 84/02913 (Rogers et al., Mosanto).

Promoters which are known or found to cause transcription of DNA inplant cells can be used in the present invention. Such promoters may beobtained from a variety of sources such as plants and plant DNA virsuesand include, but are not limited to, the CaMV35A and FMV35S promotersand promoters isolated from plant genes such as ssRUBISCO genes and themaize ubiquitin and rice actin genes. As described below, it ispreferred that the particular promoter selected should be capable ofcausing sufficient expression to result in the production of aneffective amount of a Class II EPSPS to render the plant substantiallytolerant to glyphosate herbicides. The amount of Class II EPSPS neededto induce the desired tolerance may vary with the plant species. It ispreferred that the promoters utilized have relatively high expression inall meristematic tissues in addition to other tissues inasmuch as it isnow known that glyphosate is translocated and accumulated in this typeof plant tissue. Alternatively, a combination of chimeric genes can beused to cumulatively result in the necessary overall expression level ofthe selected Class II EPSPS enzyme to result in the glyphosate-tolerantphenotype.

The mRNA produced by a DNA construct of the present invention alsocontains a 5′ non-translated leader sequence. This sequence can bederived from the promoter selected to express the gene, and can bespecifically modified so as to increase translation of the mRNA. The 5′non-translated regions can also be obtained from viral RNAs, fromsuitable eukaryotic genes, or from a synthetic gene sequence. Thepresent invention is not limited to constructs, as presented in thefollowing examples, wherein the non-translated region is derived fromboth the 5′ non-translated sequence that accompanies the promotersequence and part of the 5′ non-translated region of the virus coatprotein gene. Rather, the non-translated leader sequence can be derivedfrom an unrelated promoter or coding sequence as discussed above.

Preferred promoters for use in the present invention the full-lengthtranscript (SEQ ID NO:1) promoter from the figwort mosaic virus (FMV35S)and the full-length transcript (35S) promoter from cauliflower mosaicvirus (CaMV), including the enhanced CaMV35S promoter (Kay et al. 1987).The FMV35S promoter functions as strong and uniform promoter withparticularly good expression in meristematic tissue for chimeric genesinserted into plants, particularly dicotyledons. The resultingtransgenic plant in general expresses the protein encoded by theinserted gene at a higher and more uniform level throughout the tissuesand cells of the transformed plant than the same gene driven by anenhanced CaMV35S promoter. Referring to FIG. 1, the DNA sequence (SEQ IDNO:1) of the FMV35S promoter is located between nucleotides 6368 and6930 of the FMV genome. A 5′ non-translated leader sequence ispreferably coupled with the promoter. The leader sequence can be fromthe FMV35S genome itself or can be from a source other than FMV35S.

For expression of heterologous genes in moncotyledonous plants the useof an intron has been found to enhance expression of the heterologousgene. While one may use any of a number of introns which have beenisolated from plant genes, the use of the first intron from the maizeheat shock 70 gene is preferred.

The 3′ non-translated region of the chimeric plant gene contains apolyadenylation signal which functions in plants to cause the additionof polyadenylate nucleotides to the 3′ end of the viral RNA. Examples ofsuitable 3′ regions are (1) the 3′ transcribed, non-translated regionscontaining the polyadenylated signal of Agrobacterium tumor-inducing(Ti) plasmid genes, such as the nopaline synthase (NOS) gene, and (2)plant genes like the soybean storage protein genes and the small subunitof the ribulose-1,5-biphosphate carboxylase (ssRUBISCO) gene. An exampleof a preferred 3′ region is that from the ssRUBISCO gene from pea (E9),described in greater detail below.

The DNA constructs of the present invention also contain a structuralcoding sequence in double-stranded DNA form which encodes aglyphosate-tolerant, highly efficient Class II EPSPS enzyme.

Identification of glyphosate-tolerant, highly efficient EPSPS enzymes

In an attempt to identify and isolate glyphosate-tolerant, highlyefficient EPSPS enzymes, kinetic analysis of the EPSPS enzymes from anumber of bacteria exhibiting tolerance to glyphosate or that had beenisolated from suitable sources was undertaken. It was discovered that insome cases the EPSPS enzymes showed no tolerance to inhibition byglyphosate and it was concluded that the tolerance phenotype of thebacterium was due to an impermeability to glyphosate or other factors.In a number of cases, however, microorganisms were identified whoseEPSPS enzyme showed a greater degree of tolerance to inhibition byglyphosate and that displayed a low K_(m) for PEP when compared to thatpreviously reported for other microbial and plant sources. The EPSPSenzymes from these microorganisms were then subjected to further studyand analysis.

Table I displays the data obtained for the EPSPS enzymes identified andisolated as a result of the above described analysis. Table I includesdata for three identified Class II EPSPS enzymes that were observed tohave a high tolerance to inhibition to glyphosate and a low K_(m) forPEP as well as data for the native Petunia EPSPS and aglyphosate-tolerant variant of the Petunia EPSPS referred to as GA101.The GA101 variant is so named because it exhibits the substitution of analanine residue for a glycine residue at position 101 (with respect toPetunia). When the change introduced into the Petunia EPSPS (GA101) wasintroduced into a number of other EPSPS enzymes, similar changes in akinetics were observed, an elevation of the K_(i) for glyphosate and ofthe K_(m) for PEP.

TABLE I Kinetic characterization of EPSPS enzymes ENZYME K_(m) PEP K_(l)Glyphosate SOURCE (μM) (μM) K_(l)/K_(m) Petunia 5 0.4 0.08 Petunia GA101200 2000 10 PG2982   2.1-3.1¹ 25-82 ˜8-40 LBAA ˜7.3-8²  60 (est)⁷ ˜7.9CP4 12³ 2720 227 B. subtilis 1A2 13⁴ 440 33.8 S. aureus 5⁵ 200 40 ¹Rangeof PEP tested = 1-40 μM ²Range of PEP tested = 5-80 μM ³Range of PEPtested = 1.5-40 μM ⁴Range of PEP tested = 1-60 μM ⁵Range of PEP tested =1-50 μM ⁷(est) = estimated

The Agrobacterium sp. strain CP4 was initially identified by its abilityto grow on glyphosate as a carbon source (10 mM) in the presence of 1 mMphosphate. The strain CP4 was identified from a collection obtained froma fixed-bed immobilized cell column that employed Mannville R-635diatomaceous earth beads. The column had been run for three months on awaste-water feed from a glyphosate production plant. The columncontained 50 mg/ml glyphosate and NH₃ as NH₄Cl. Total organic carbon was300 mg/ml and BOD's (Biological Oxygen Demand—a measure of “soft” carbonavailability) were less than 30 mg/ml. This treatment column has beendescribed (Heitkamp et al., 1990). Dworkin-Foster minimal salts mediumcontaining glyphosate at 10 mM and with phosphate at 1 mM was used toselect for microbes from a wash of this column that were capable ofgrowing on glyphosate as sole carbon source. Dworkin-Foster minimalmedium was made up by combining in I liter (with autoclaved H₂O), 1 mleach of A, B and C and 10 ml of D (as per below) and thiamine HCl (5mg).

A. D-F Salts (1000X stock; per 100 ml; autoclaved): H₂BO₃ 1 mg MnSO₄.7H₂O 1 mg ZnSO₄.7 H₂O 12.5 mg CuSO₄.5 H₂O 8 mg NaMoO₃.3 H₂O 1.7 mg B.FeSO₄.7 H₂O (1000X Stock; per 100 0.1 g ml; autoclaved) C. MgSO₄.7 H₂O(1000X Stock; per 100 20 g ml; autoclaved) D. (NH₄)₂SO₄ (100X stock; per100 ml; 20 g autoclaved)

Yeast Extract (YE; Difco) was added to a final concentration of 0.01 or0.001%. The strain CP4 was also grown on media composed of D-F salts,amended as described above, containing glucose, gluconate and citrate(each at 0.1%) as carbon sources and with inorganic phosphate (0.2-1.0mM) as the phosphorous source.

Other Class II EPSPS containing microorganisms were identified asAchromobacter sp. strain LBAA (Hallas et al., 1988), Pseudomonas sp.strain PG2982 (Moore et al., 1983; Fitzgibbon 1988), Bacillus subtilis1A2 (Henner et al., 1984) and Staphylococcus aureus (O'Connell et al.,1993). It had been reported previously, from measurements in crudelysates, that the EPSPS enzyme from strain PG2982 was less sensitive toinhibition to glyphosate than that of E. coli, but there has been noreport of the details of this lack of sensitivity and there has been noreport on the K_(m) for PEP for this enzyme or of the DNA sequence forthe gene for this enzyme (Fitzgibbon, 1988; Fitzgibbon and Braymer,1990). Relationship of the Class II EPSPS to those previously studied.

All EPSPS proteins studied to date have shown a remarkable degree ofhomology. For example, bacterial and plant EPSPS's are about 54%identical and with similarity as high as 80%. Within bacterial EPSPS'sand plant EPSPS's themselves the degree of identity and similarity ismuch greater (see Table II).

TABLE II Comparison between exemplary Class I EPSPS protein sequences¹similarity identity E. coli vs. S. typhaurium 93 88 P. hybrids vs. E.coli 72 55 P. hybrids vs. L. excalentum 93 88 ¹The EPSPS sequencescompared here were obtained from the following reference: E. coli,Rogers et al., 1983; S. typhourium, Smetzer et al, 1985; Petanoichybrids; Shah et al, 1986; and tomato (L. escalautum), Gasper et al,1988.

When crude extracts of CP4 and LBAA bacteria (50 μg protein) were probedusing rabbit anti-EPSPS antibody (Padgette et al., 1987) to the PetuniaEPSPS protein in a Western analysis, no positive signal could bedetected, even with extended exposure times (Protein A—¹²⁵I developmentsystem) and under conditions where the control EPSPS (Petunia EPSPS, 20ng; a Class I EPSPS) was readily detected. The presence of EPSPSactivity in these extracts was confirmed by enzyme assay. Thissurprising result, indicating a lack of similarity between the EPSPS'sfrom these bacterial isolates and those previously studied, coupled withthe combination of a low K_(m) for PEP and a high K_(i) for glyphosate,illustrates that these new EPSPS enzymes are different from known EPSPSenzymes (now referred to as Class I EPSPS).

Glyphosate-tolerant Enzymes is Microbial Isolates

For clarity and brevity of disclosure, the following description of theisolation of genes encoding Class II EPSPS enzymes is directed to theisolation of such a gene from a bacterial isolate. Those skilled in theart will recognize that the same or similar strategy can be utilized toisolate such genes from other microbial isolates, plant or fungalsources.

Cloning of the Agrobacterium sp. strain CP4 EPSPS Gene(s) in E. coli

Having established the existence of a suitable EPSPS in Agrobacteriumsp. strain CP4, two parallel approaches were undertaken to clone thegene: cloning based on the expected phenotype for a glyphosate-tolerantEPSPS; and purification of the enzyme to provide material to raiseantibodies and to obtain amino acid sequences from the protein tofacilitate the verification of clones. Cloning and genetic techniques,unless otherwise indicated, are generally those described in Maniatis etal., 1982 or Sambrook et al., 1987. The cloning strategy was as follows:introduction of a cosmid bank of strain Agrobacterium sp. strain CP4into E. coli and selection for the EPSPS gene by selection for growth oninhibitory concentrations of glyphosate.

Chromosomal DNA was prepared from strain Agrobacterium sp. strain CP4 asfollows: The cell pellet from a 200 ml L-Broth (Miller, 1972), late logphase culture of Agrobacterium sp. strain CP4 was resuspended in 10 mlof Solution I; 50 mM Glucose, 10 mM EDTA, 25 mM Tris-CL pH 8.0 (Birnboimand Doly, 1979). SDS was added to a final concentration of 1% and thesuspension was subjected to three freeze-thaw cycles, each consisting ofimmersion in dry ice for 15 minutes and in water at 70° C. for 10minutes. The lysate was then extracted four times with equal volumes ofphenol:chloroform (1:1; phenol saturated with TE; TE=10 mM Tris pH8.0;1.0 mM EDTA) and the phases separated by centrifugation (15000 g; 10minutes). The ethanol-precipitable material was pelleted from thesupernatant by brief centrifugation (8000 g; 5 minutes) followingaddition of two volumes of ethanol. The pellet was resuspended in 5 mlTE and dialyzed for 16 hours at 4° C. against 2 liters TE. Thispreparation yielded a 5 ml DNA solution of 552 μg/ml.

Partially-restricted DNA was prepared as follows. Three 100 μg aliquotsamples of CP4 DNA were treated for 1 hour at 37° C. with restrictionendonuclease HindIII at rates of 4, 2 and 1 enzyme unit/μg DNA,respectively. The DNA samples were pooled, made 0.25 mM with EDTA andextracted with an equal volume of phenol:chloroform. Following theaddition of sodium acetate and ethanol, the DNA was precipitated withtwo volumes of ethanol and pelleted by centrifugation (12000 g; 10minutes). The dried DNA pellet was resuspended in 500 μl TE and layeredon a 10-40% Sucrose gradient (in 5% increments of 5.5 ml each) in 0.5MNaCl, 50 mM Tris pH8.0, 5 mM EDTA. Following centrifugation for 20 hoursat 26,000 rpm in a SW28 rotor, the tubes were punctured and ˜1.5 mlfractions collected. Samples (20 μl) of each second fraction were run on0.7% agarose gel and the size of the DNA determined by comparison withlinearized lambda DNA and HindIII-digested lambda DNA standards.Fractions containing DNA of 25-35 kb fragments were pooled, desalted onAMICON10 columns (7000 rpm; 20° C.; 45 minutes) and concentrated byprecipitation. This procedure yielded 15 μg of CP4 DNA of the requiredsize. A cosmid bank was constructed using the vector pMON17020. Thisvector, a map of which is presented in FIG. 2, is based on the pBR327replicon and contains the spectinomycin/streptomycin (Sp^(r);spc)resistance gene from Tn7 (Fling et al., 1985), the chloramphenicolresistance gene (Cm^(r);cat) from Tn9 (Alton et al., 1979), the gene10promoter region from phage T7 (Dunn et al., 1983), and the 1.6 kb BglIIphage lambda cos fragment from pHC79 (Hohn and Collins, 1980). A numberof cloning sites are located downstream of the cat gene. Since thepredominant block to the expression of genes from other microbialsources in E. coli appears to be at the level of transcription, the useof the T7 promoter and supplying the T7 polymerase in trans from thepGP1-2 plasmid (Tabor and Richardson, 1985), enables the expression oflarge DNA segments of foreign DNA, even those containing RNA polymerasetranscription termination sequences. The expression of the spc gene isimpaired by transcription from the T7 promoter such that only Cmr can beselected in strains containing pGP1-2. The use of antibiotic resistancessuch as Cm resistance which do not employ a membrane component ispreferred due to the observation that high level expression ofresistance genes that involve a membrane component, i.e. β-lactamase andAmp resistance, give rise to a glyphosate-tolerant phenotype.Presumably, this is due to the exclusion of glyphosate from the cell bythe membrane localized resistance protein. It is also preferred that theselectable marker be oriented in the same direction as the T7 promoter.

The vector was then cut with HindIII and treated with calf alkalinephosphatase (CAP) in preparation for cloning. Vector and targetsequences were ligated by combining the following:

Vector DNA (HindIII/CAP) 3 μg Size fractionated CP4 HindIII fragments1.5 μg 10X ligation buffer 2.2 μl T4 DNA ligase (New England Biolabs)(400 U/μl) 1.0 μland adding H₂O to 22.0 μl. This mixture was incubated for 18 hours at16° C. 10X ligation buffer is 250 mM Tris-HCl, pH 8.0; 100 mM MgCl₂; 100mM Dithiothreitol; 2 mM Spermidine. The ligated DNA (5 μl) was packagedinto lambda phage particles (Stratagene; Gigapack Gold) using themanufacturer's procedure.

A sample (200 μl) of E. coli HB101 (Boyer and Rolland-Dussoix, 1973)containing the T7 polymerase expression plasmid pGP1-2 (Tabor andRichardson, 1985) and grown overnight in L-Broth (with maltose at 0.2%and kanamycin at 50/μg/ml) was infected with 50 μl of the packaged DNA.Transformants were selected at 30° C. on M9 (Miller, 1972) agarcontaining kanamycin (50 μg/ml), chloramphenicol (25 μg/ml), L-proline(50 μg/ml), L-leucine (50 μg/ml) and B1 (5 μg/ml), and with glyphosateat 3.0 mM. Aliquot samples were also plated on the same media lackingglyphosate to titer the packaged cosmids. Cosmid transformants wereisolated on this latter medium at a rate of ˜5×10⁵ per μg CP4 HindIIIDNA after 3 days at 30° C. Colonies arose on the glyphosate agar fromday 3 until day 15 with a final rate of ˜1 per 200 cosmids. DNA wasprepared from 14 glyphosate-tolerant clones and, following verificationof this phenotype, was transformed into E. coli GB100/pGP1-2 (E. coliGB100 is an aroA derivative of MM294 [Talmadge and Gilbert, 1980]) andtested for complementation for growth in the absence of added aromaticamino acids and aminobenzoic acids. Other aroA strains such as SR481(Bachman et al., 1980; Padgette et al., 1987), could be used and wouldbe suitable for this experiment. The use of GB100 is merely exemplaryand should not be viewed in a limiting sense. This aroA strain usuallyrequires that growth media be supplemented with L-phenylalanine,L-tyrosine and L-tryptophan each at 100 μg/ml and withpara-hydroxybenzoic acid, 2,3-dihydroxybenzoic acid andpara-aminobenzoic acid each at 5 μg/ml for growth in minimal media. Ofthe fourteen cosmids tested only one showed complementation of the aroA-phenotype. Transformants of this cosmid, pMON17076, showed weak butuniform growth on the unsupplemented minimal media after 10 days.

The proteins encoded by the cosmids were determined in vivo using a T7expression system (Tabor and Richardson, 1985). Cultures of E. colicontaining pGP1-2 (Tabor and Richardson, 1985) and test and controlcosmids were grown at 30° C. in L-broth (2 ml) with chloramphenicol andkanamycin (25 and 50 μg/ml, respectively) to a Klett reading of ˜50. Analiquot was removed and the cells collected by centrifugation, washedwith M9 salts (Miller, 1972) and resuspended in 1 ml M9 mediumcontaining glucose at 0.2%, thiamine at 20 μg/ml and containing the 18amino acids at 0.01% (minus cysteine and methionine). Followingincubation at 30° C. for 90 minutes, the cultures were transferred to a42° C. water bath and held there for 15 minutes. Rifampicin (Sigma) wasadded to 200 μg/ml and the cultures held at 42° C. for 10 additionalminutes and then transferred to 30° C. for 20 minutes. Samples werepulsed with 10 μCi of ³⁵S-methionine for 5 minutes at 30° C. The cellswere collected by centrifugation and suspended in 60-120 μl crackingbuffer (60 mM Tris-HCl 6.8, 1% SDS, 1% 2-mercaptoethanol, 10% glycerol,0.01% bromophenol blue). Aliquot samples were electrophoresed on 12.5%SDS-PAGE and following soaking for 60 minutes in 10 volumes of AceticAcid-Methanol-water (10:30:60), the gel was soaked in ENLIGHTNING™(DUPONT) following manufacturer's directions, dried, and exposed at −70°C. to X-Ray film. Proteins of about 45 kd in size, labeled with³⁵S-methionine, were detected in number of the cosmids, includingpMON17076.

Purification of EPSPS from Agrobacterium sp. strain CP4

All protein purification procedures were carried out at 3°-5° C. EPSPSenzyme assays were performed using either the phosphate release orradioactive HPLC method, as previously described in Padgette et al.,1987, using 1 mM phosphoenol pyruvate (PEP, Boehringer) and 2 mMshikimate-3-phosphate (S3P) substrate concentrations. For radioactiveHPLC assays, ¹⁴-CPEP (Amersham) was utilized. S3P was synthesized aspreviously described in Wibbenmeyer et al. 1988. N-terminal amino acidsequencing was performed by loading samples onto a Polybrene precycledfilter in aliquots while drying. Automated Edman degradation chemistrywas used to determine the N-terminal protein sequence, using an AppliedBiosystems Model 470A gas phase sequencer (Hunkapiller et al., 1983)with an Applied Biosystems 120A PTH analyzer.

Five 10-liter fermentations were carried out on a spontaneous “smooth”isolate of strain CP4 that displayed less clumping when grown in liquidculture. This reduced clumping and smooth colony morphology may be dueto reduced polysaccharide production by this isolate. In the followingsection dealing with the purification of the EPSPS enzyme, CP4 refers tothe “smooth” isolate—CP4-S1. The cells from the three batches showingthe highest specific activities were pooled. Cell paste of Agrobacteriumsp. CP4 (300 g) was washed twice with 0.5 L of 0.9% saline and collectedby centrifugation (30 minutes, 8000 rpm in a GS3 Sorvall rotor). Thecell pellet was suspended in 0.9 L extraction buffer (100 mM TrisCl, 1mM EDTA, 1 mM BAM (Benzamidine), 5 mM DTT, 10% glycerol, pH 7.5) andlysed by 2 passes through a Manton Gaulin cell. The resulting solutionwas centrifuged (30 minutes, 8000 rpm) and the supernatant was treatedwith 0.21 L of 1.5% protamine sulfate (in 100 mM TrisCl, pH 7.5, 0.2%w/v final protamine sulfate concentration). After stirring for 1 hour,the mixture was centrifuged (50 minutes, 8000 rpm) and the resultingsupernatant treated with solid ammonium sulfate to 40% saturation andstirred for 1 hour. After centrifugation (50 minutes, 8000 rpm), theresulting supernatant was treated with solid ammonium sulfate to 70%saturation, stirred for 50 minutes, and the insoluble protein wascollected by centrifugation (1 hour, 8000 rpm). This 40-70% ammoniumsulfate fraction was then dissolved in extraction buffer to give a finalvolume of 0.2 L, and dialyzed twice (Spectrum 10,000 MW cutoff dialysistubing) against 2 L of extraction buffer for a total of 12 hours.

To the resulting dialyzed 40-70% ammonium sulfate fraction (0.29 L) wasadded solid ammonium sulfate to give a final concentration of 1M. Thismaterial was loaded (2 ml/min) onto a column (5 cm×15 cm, 295 ml) packedwith phenyl Sepharose CL-4B (Pharmacia) resin equilibrated withextraction buffer containing 1M ammonium sulfate, and washed with thesame buffer (1.5 L, 2 ml/min). EPSPS was eluted with a linear gradientof extraction buffer going from 1M to 0.00M ammonium sulfate (totalvolume of 1.5 L, 2 ml/min). Fractions were collected (20 ml) and assayedfor EPSPS activity by the phosphate release assay. The fractions withthe highest EPSPS activity (fractions 36-50) were pooled and dialyzedagainst 3×2 L (18 hours) of 10 mM TrisCl, 25 mM KCl, 1 mM EDTA, 5 mMDTT, 10% glycerol, pH 7.8.

The dialyzed EPSPS extract (350 ml) was loaded (5 ml/min) onto a column(2.4 cm×30 cm, 136 ml) packed with Q-Sepharose Fast Flow (Pharmacia)resin equilibrated with 10 mM TrisCl, 25 mM KCl, 5 mM DTT, 10% glycerol,pH 7.8 (Q Sepharose buffer), and washed with 1 L of the same buffer.EPSPS was eluted with a linear gradient of Q Sepharose buffer going from0.025M to 0.40M KCl (total volume of 1.4 L, 5 ml/min). Fractions werecollected (15 ml) and assayed for EPSPS activity by the phosphaterelease assay. The fractions with the highest EPSPS activity (fractions47-60) were pooled and the protein was precipitated by adding solidammonium sulfate to 80% saturation and stirring for 1 hour. Theprecipitated protein was collected by centrifugation (20 minutes, 12000rpm in a GSA Sorvall rotor), dissolved in Q Sepharose buffer (totalvolume of 14 ml), and dialyzed against the same buffer (2×1 L, 18hours).

The resulting dialyzed partially purified EPSPS extract (19 ml) wasloaded (1.7 ml/min) onto a Mono Q 10/10 column (Pharmacia) equilibratedwith Q Sepharose buffer, and washed with the same buffer (35 ml). EPSPSwas eluted with a linear gradient of 0.025M to 0.35M KCl (total volumeof 119 ml, 1.7 ml/min). Fractions were collected (1.7 ml) and assayedfor EPSPS activity by the phosphate release assayed. The fractions withthe highest EPSPS activity (fractions 30-37) were pooled (6 ml).

The Mono Q pool was made 1M in ammonium sulfate by the addition of solidammonium sulfate and 2 ml aliquots were chromatographed on a PhenylSuperose 5/5 column (Pharmacia) equilibrated with 100 mM TrisCl, 5 mMDTT, 1M ammonium sulfate, 10% glycerol, pH 7.5 (Phenyl Superose buffer).Samples were loaded (1 ml/min), washed with Phenyl Superose buffer (10ml), and eluted with a linear gradient of Phenyl Superose buffer goingfrom 1M to 0.00M ammonium sulfate (total volume of 60 ml, 1 ml/min).Fractions were collected (1 ml) and assayed for EPSPS activity by thephosphate release assay. The fractions from each run with the highestEPSPS activity (fractions ˜36-40) were pooled together (10 ml, 2.5 mgprotein). For N-terminal amino acid sequence determination, a portion ofone fraction (#39 from run 1) was dialyzed against 50 mM NaHCO₃ (2×1 L).The resulting pure EPSPS sample (0.9 ml, 77 μg protein) was found toexhibit a single N-terminal amino acid sequence of:XH(G)ASSRPATARKSS(G)LX(G)(T)V(R)IPG(D)(K)(M) (SEQ ID NO:18).

The remaining Phenyl Superose EPSPS pool was dialyzed against 50 mMTrisCl, 2 mM DTT, 10 mM KCl, 10% glycerol, pH 7.5 (2×1 L). An aliquot(0.55 ml, 0.61 mg protein) was loaded (1 ml/min) onto a Mono Q 5/5column (Pharmacia) equilibrated with Q Sepharose buffer, washed with thesame buffer (5 ml), and eluted with a linear gradient of Q Sepharosebuffer going from 0-0.14M KCl in 10 minutes, then holding at 0.14M KCl(1 ml/min). Fractions were collected (1 ml) and assayed for EPSPSactivity by the phosphate release assay and were subjected to SDS-PAGE(10-15%, Phast System, Pharmacia, with silver staining) to determineprotein purity. Fractions exhibiting a single band of protein bySDS-PAGE (22-25, 222 μg) were pooled and dialyzed against 100 mMammonium bicarbonate, pH 8.1 (2×1 L, 9 hours).

Trypsinolysis and peptide sequencing of Agrobacterium sp strain CP4EPSPS

To the resulting pure Agrobacterium sp. strain CP4 EPSPS (111 μg) wasadded 3 μg of trypsin (Calbiochem), and the trypsinolysis reaction wasallowed to proceed for 16 hours at 37° C. The tryptic digest was thenchromatographed (1 ml/min) on a C18 reverse phase HPLC column (Vydac) aspreviously described in Padgette et al., 1988 for E. coli EPSPS. For allpeptide purifications, 0.1% trifluoroacetic acid (TFA, Pierce) wasdesignated buffer “RP-A” and 0.1% TFA in acetonitrile was buffer “RP-B”.The gradient used for elution of the trypsinized Agrobacterium sp. CP4EPSPS was: 0-8 minutes, 0% RP-B; 8-28 minutes, 0-15% RP-B; 28-40minutes, 15-21% RP-B; 40-68 minutes, 21-49% RP-B; 68-72 minutes, 49-75%RP-B; 72-74 minutes, 75-100% RP-B. Fractions were collected (1 ml) and,based on the elution profile at 210 nm, at least 70 distinct peptideswere produced from the trypsinized EPSPS. Fractions 40-70 wereevaporated to dryness and redissolved in 150 μl each of 10%acetonitrile, 0.1% trifluoroacetic acid.

The fraction 61 peptide was further purified on the C18 column by thegradient: 0-5 minutes, 0% RP-B; 5-10 minutes, 0-38% RP-B; 10-30 minutes,38-45% B. Fractions were collected based on the UV signal at 210 nm. Alarge peptide peak in fraction 24 eluted at 42% RP-B and was dried down,resuspended as described above, and rechromatographed on the C18 columnwith the gradient: 0-5 minutes, 0% RP-B; 5-12 min, 0-38% RP-B; 12-15min, 38-39% RP-B; 15-18 minutes, 39% RP-B; 18-20 minutes, 39-41% RP-B;20-24 minutes, 41% RP-B; 24-28 minutes, 42% RP-B. The peptide infraction 25, eluting at 41% RP-B and designated peptide 61-24-25, wassubjected to N-terminal amino acid sequencing, and the followingsequence was determined:APSM(I)(D)EYPILAV (SEQ ID NO:19)The CP4 EPSPS fraction 53 tryptic peptide was further purified by C18HPLC by the gradient 0% B (5 minutes), 0-30% B (5-17 minutes), 30-40% B(17-37 minutes). The peptide in fraction 28, eluting at 34% B anddesignated peptide 53-28, was subjected to N-terminal amino acidsequencing, and the following sequence was determined:ITGLLEGEDVINTGK (SEQ ID NO:20).

In order to verify the CP4 EPSPS cosmid clone, a number ofoligonucleotide probes were designed on the basis of the sequence of twoof the tryptic sequences from the CP4 enzyme (Table III). The probeidentified as MID was very low degeneracy and was used for initialscreening. The probes identified as EDV-C and EDV-T were based on thesame amino acid sequences and differ in one position (underlined inTable III below) and were used as confirmatory probes, with a positiveto be expected only from one of these two probes. In theoligonucleotides below, alternate acceptable nucleotides at a particularposition are designated by a “/” such as A/C/T.

TABLE III Selected CP4 EPSPS peptide sequences and DNA probes PEPTIDE61-24-25 APSM(I)(D)EYPILAV (SEQ ID NO:19) Probe MID; 17-mer; mixedprobe; 24-fold degenerate (SEQ ID NO:21) ATGATA/C/TGAC/TGAG/ATAC/TCCPEPTIDE 53-28 ITGLLEGEDVINTGK (SEQ ID NO:20) Probe EDV-C; 17-mer; mixedprobe; 48-fold (SEQ ID NO:22) degenerate GAA/GGAC/TGTA/C/G/TATA/C/TAACACProbe EDV-T; 17-mer; mixed probe; 48-fold (SEQ ID NO:23) degenerateGAA/GGAC/TGTA/C/G/TATA/C/TAATAC

The probes were labeled using gamma-³²P-ATP and polynucleotide kinase.DNA from fourteen of the cosmids described above was restricted withEcoRI, transferred to membrane and probed with the oligonucleotideprobes. The conditions used were as follows: prehybridization wascarried out in 6× SSC, 10× Denhardt's for 2-18 hour periods at 60° C.,and hybridization was for 48-72 hours in 6× SSC, 10× Denhardt's, 100μg/ml tRNA at 10° C. below the T_(d) for the probe. The T_(d) of theprobe was approximated by the formula 2° C×(A+T)+4° C×(G+C). The filterswere then washed three times with 6× SSC for ten minutes each at roomtemperature, dried and autoradiographed. Using the MID probe, an ˜9.9 kbfragment in the pMON17076 cosmid gave the only positive signal. Thiscosmid DNA was then probed with the EDV-C (SEQ ID NO:22) and EDV-T (SEQID NO:23) probes separately and again this ˜9.9 kb band gave a signaland only with the EDV-T probe.

The combined data on the glyphosate-tolerant phenotype, thecomplementation of the E. coli aroA- phenotype, the expression of a ˜45Kd protein, and the hybridization to two probes derived from the CP4EPSPS amino acid sequence strongly suggested that the pMON17076 cosmidcontained the EPSPS gene.

Localization and subcloning of the CP4 EPSPS gene

The CP4 EPSPS gene was further localized as follows: a number ofadditional Southern analyses were carried out on different restrictiondigests of pMON17076 using the MID (SEQ ID NO:21) and EDV-T (SEQ IDNO:23) probes separately. Based on these analyses and on subsequentdetailed restriction mapping of the pBlueScript (Stratagene) subclonesof the ˜9.9 kb fragment from pMON17076, a 3.8 kb EcoRI-SalI fragment wasidentified to which both probes hybridized. This analysis also showedthat MID (SEQ ID NO:21) and EDV-T (SEQ ID NO:23) probes hybridized todifferent sides of BamHI, ClaI, and SacII sites. This 3.8 kb fragmentwas cloned in both orientations in pBlueScript to form pMON17081 andpMON17082. The phenotypes imparted to E. coli by these clones were thendetermined. Glyphosate tolerance was determined following transformationinto E. coli MM294 containing pGP1-2 (pBlueScript also contains a T7promoter) on M9 agar media containing glyphosate at 3 mM. Both pMON17081and pMON17082 showed glyphosate-tolerant colonies at three days at 30°C. at about half the size of the controls on the same media lackingglyphosate. This result suggested that the 3.8 kb fragment contained anintact EPSPS gene. The apparent lack of orientation-dependence of thisphenotype could be explained by the presence of the T7 promoter at oneside of the cloning sites and the lac promoter at the other. The aroAphenotype was determined in transformants of E. coli GB100 on M9 agarmedia lacking aromatic supplements. In this experiment, carried out withand without the Plac inducer IPTG, pMON17082 showed much greater growththan pMON17081, suggesting that the EPSPS gene was expressed from theSalI site towards the EcoRI site.

Nucleotide sequencing was begun from a number of restriction site ends,including the BamHI site discussed above. Sequences encoding proteinsequences that closely matched the N-terminus protein sequence and thatfor the tryptic fragment 53-28 (SEQ ID NO:20) (the basis of the EDV-Tprobe) (SEQ ID NO:23) were localized to the SalI side of this BamHIsite. These data provided conclusive evidence for the cloning of the CP4EPSPS gene and for the direction of transcription of this gene. Thesedata coupled with the restriction mapping data also indicated that thecomplete gene was located on an ˜2.3 kb XhoI fragment and this fragmentwas subcloned into pBlueScript. The nucleotide sequence of almost 2 kbof this fragment was determined by a combination of sequencing fromcloned restriction fragments and by the use of specific primers toextend the sequence. The nucleotide sequence of the CP4 EPSPS gene andflanking regions is shown in FIG. 3 (SEQ ID NO:2). The sequencecorresponding to peptide 61-24-25 (SEQ ID NO:19) was also located. Thesequence was determined using both the SEQUENASE™ kit from IBI(International Biotechnologies Inc.) and the T7 sequencing/Deaza Kitfrom Pharmacia.

That the cloned gene encoded the EPSPS activity purified from theAgrobacterium sp. strain CP4 was verified in the following manner: By aseries of site directed mutageneses, BglII and NcoI sites were placed atthe N-terminus with the fMet contained within the NcoI recognitionsequence, the first internal NcoI site was removed (the second internalNcoI site was removed later), and a SacI site was placed after the stopcodons. At a later stage the internal NotI site was also removed bysite-directed mutagenesis. The following list includes the primers forthe site-directed mutagenesis (addition or removal of restriction sites)of the CP4 EPSPS gene. Mutagenesis was carried out by the procedures ofKunkel et al. (1987), essentially as described in Sambrook et al.(1989).

PRIMER BgNc (addition of BgIII and NcoI sites to N-terminus)CGTGGATAGATCTAGGAAGACAACCATGGCTCACGGTC (SEQ ID NO:24) PRIMER Sph2(addition of SphI site to N-terminus)GGATAGATTAAGGAAGACGCGCATGCTTCACGGTGCAAGCAGCC (SEQ ID NO:25) PRIMER S1(addition of SacI site immediately after stop codons)GGCTGCCTGATGAGCTCCACAATCGCCATCGATGG (SEQ ID NO:26) PRIMER N1 (removal ofinternal NotI recognition site) CGTCGCTCGTCGTGCGTGGCCGCCCTGACGGC (SEQ IDNO:27) PRIMER NcoI (removal of first internal NcoI recognition site)CGGGCAAGGCCATGCAGGCTATGGGCGCC (SEQ ID NO:28) PRIMER Nco2 (removal ofsecond internal NcoI recognition site) CGGGCTGCCGCCTGACTATGGGCCTCGTCGG(SEQ ID NO:29)

This CP4 EPSPS gene was then cloned as a NcoI-BamHI N-terminal fragmentplus a BamHI-SacI C-terminal fragment into a PrecA-gene10L expressionvector similar to those described (Wong et al., 1988; Olins et al.,1988) to form pMON17101. The K_(m) for PEP and the K_(i) for glyphosatewere determined for the EPSPS activity in crude lysates ofpMON17101/GB100 transformants following induction with nalidixic acid(Wong et al., 1988) and found to be the same as that determined for thepurified and crude enzyme preparations from Agrobacterium sp. strainCP4.

Characterization of the EPSPS gene from Achromobacter sp. strain LBAAand from Pseudomonas sp. strain PG2982

A cosmid bank of partially HindIII-restricted LBAA DNA was constructedin E. coli MM294 in the vector pHC79 (Hohn and Collins, 1980). This bankwas probed with a full length CP4 EPSPS gene probe by colonyhybridization and positive clones were identified at a rate of ˜1 per400 cosmids. The LBAA EPSPS gene was further localized in these cosmidsby Southern analysis. The gene was located on an ˜2.8 kb XhoI fragmentand by a series of sequencing steps, both from restriction fragment endsand by using the oligonucleotide primers from the sequencing of the CP4EPSPS gene, the nucleotide sequence of the LBAA EPSPS gene was completedand is presented in FIG. 4 (SEQ ID NO:4).

The EPSPS gene from PG2982 was also cloned. The EPSPS protein waspurified, essentially as described for the CP4 enzyme, with thefollowing differences: Following the Sepharose CL-4B column, thefractions with the highest EPSPS activity were pooled and the proteinprecipitated by adding solid ammonium sulfate to 85% saturation andstirring for 1 hour. The precipitated protein was collected bycentrifugation, resuspended in Q Sepharose buffer and following dialysisagainst the same buffer was loaded onto the column (as for the CP4enzyme). After purification on the Q Sepharose column, ˜40 mg of proteinin 100 mM Tris pH 7.8, 10% glycerol, 1 mM EDTA, 1 mM DTT, and 1Mammonium sulfate, was loaded onto a Phenyl Superose (Pharmacia) column.The column was eluted at 1.0 ml/minutes with a 40 ml gradient from 1.0Mto 0.00M ammonium sulfate in the above buffer.

Approximately 1.0 mg of protein from the active fractions of the PhenylSuperose 10/10 column was loaded onto a Pharmacia Mono P 5/10Chromatofocusing column with a flow rate of 0.75 ml/minutes. Thestarting buffer was 25 mM bis-Tris at pH 6.3, and the column was elutedwith 39 ml of Polybuffer 74, pH 4.0. Approximately 50 μg of the peakfraction from the Chromatofocusing column was dialyzed into 25 mMammonium bicarbonate. This sample was then used to determine theN-terminal amino acid sequence.

The N-terminal sequence obtained was:XHSASPKPATARRSE (where X=an unidentified residue) (SEQ ID NO:30)

A number of degenerate oligonucleotide probes were designed based onthis sequence and used to probe a library of PG2982 partial-HindIII DNAin the cosmid pHC79 (Hohn and Collins, 1980) by colony hybridizationunder nonstringent conditions. Final washing conditions were 15 minuteswith 1× SSC, 0.1% SDS at 55° C. One probe with the sequenceGCGGTBGCSGGYTTSGG (where B=C, G, or T; S=C or G, and Y=C or T) (SEQ IDNO:31) identified a set of cosmid clones.

The cosmid set identified in this way was made up of cosmids of diverseHindIII fragments. However, when this set was probed with the CP4 EPSPSgene probe, a cosmid containing the PG2982 EPSPS gene was identified(designated as cosmid 9C1 originally and later as pMON20107). By aseries of restriction mappings and Southern analysis this gene waslocalized to a ˜2.8 kb XhoI fragment and the nucleotide sequence of thisgene was determined. This DNA sequence (SEQ ID NO:6) is shown in FIG. 5.There are no nucleotide differences between the EPSPS gene sequencesfrom LBAA (SEQ ID NO:4) and PG2982 (SEQ ID NO:6). The kinetic parametersof the two enzymes are within the range of experimental error.

A gene from PG2982 that imparts glyphosate tolerance in E. coli has beensequenced (Fitzgibbon, 1988; Fitzgibbon and Brayruer, 1990). Thesequence of the PG2982 EPSPS Class II gene shows no homology to thepreviously reported sequence suggesting that the glyphosate-tolerantphenotype of the previous work is not related to EPSPS.

Characterization of the EPSPS from Bacillus subtilis

Bacillus subtilis 1A2 (prototroph) was obtained from the BacillusGenetic Stock Center at Ohio State University. Standard EPSPS assayreactions contained crude bacterial extract with, 1 mMphosphoenolpyruvate (PEP), 2 mM shikimate-3-phosphate (S3P), 0.1 mMammonium molybdate, 5 mM potassium fluoride, and 50 mM HEPES, pH 7.0 at25° C. One unit (U) of EPSPS activity is defined as one μmol EPSP formedper minute under these conditions. For kinetic determinations, reactionscontained crude bacterial, 2 mM S3P, varying concentrations of PEP, and50 mM HEPES, pH 7.0 at 25° C. The EPSPS specific activity was found tobe 0.003 U/mg. When the assays were performed in the presence of 1 mMglyphosphate, 100% of the EPSPS activity was retained. The appK_(m)(PEP)of the B. subtilis EPSPS was determined by measuring the reactionvelocity at varying concentrations of PEP. The results were analyzedgraphically by the hyperbolic, Lineweaver-Burk and Eadie-Hofstee plots,which yielded appK_(m)(PEP) values of 15.3 μM, 10.8 μM and 12.2 μM,respectively. These three data treatments are in good agreement, andyield an average value for appK_(m)(PEP) of 13 μM. TheappK_(i)(glyphosate) was estimated by determining the reaction rates ofB. subtilis 1A2 EPSPS in the presence of several concentrations ofglyphosphate, at a PEP concentration of 2 μM. These results werecompared to the calculated V_(max) of the EPSPS, and making theassumption that glyphosate is a competitive inhibitor versus PEP for B.subtilis EPSPS, as it is for all other characterized EPSPSs, anappK_(i)(glyphosate) was determined graphically. TheappK_(i)(glyphosate) was found to be 0.44 mM.

The EPSPS expressed from the B. subtilis aroE gene described by Henneret al. (1986) was also studied. The source of the B. subtilis aroE(EPSPS) gene was the E. coli plasmid-bearing strain ECE13 (originalcode=MM294[p trp100]; Henner, et al., 1984; obtained from the BacillusGenetic Stock Center at Ohio State University; the culture genotype is[pBR322 trp100] Ap [in MM294] [pBR322::6 kb insert with trpFBA-hisH]).Two strategies were taken to express the enzyme in E. coli GB100(aroA-): 1) the gene was isolated by PCR and cloned into anoverexpression vector, and 2) the gene was subcloned into anoverexpression vector. For the PCR cloning of the B. subtilis aroE fromECE13, two oligonucleotides were synthesized which incorporated tworestriction enzyme recognition sites (NdeI and EcoRI) to the sequencesof the following oligonucleotides:

(SEQ ID NO:45) GGAACATATGAAACGAGATAAGGTGCAG (SEQ ID NO:46)GGAATTCAAACTTCAGGATCTTGAGATAGAAAATGThe other approach to the isolation of the B. subtilis aroE gene,subcloning from ECE13 into pUC118, was performed as follows:

-   -   (i) Cut ECE13 and pUC with XmaI and SphI.    -   (ii) Isolate 1700bp aroE fragment and 2600bp pUC118 vector        fragment.    -   (iii) Ligate fragments and transform into GB100.        The subclone was designated pMON21133 and the PCR-derived clone        was named pMON21132. Clones from both approaches were first        confirmed for complementation of the aroA mutation in E. coli        GB100. The cultures exhibited EPSPS specific activities of 0.044        U/mg and 0.71 U/mg for the subclone (pMON21133) and PCR-derived        clone (pMON21132) enzymes, respectively. These specific        activities reflect the expected types of expression levels of        the two vectors. The B. subtilis EPSPS was found to be 88% and        100% resistant to inhibition by 1 mM glyphosate under these        conditions for the subcloned (pMON21133) and PCR-derived        (pMON21132) enzymes, respectively. The appK_(m) (PEP) and the        appK_(i)(glyphosate) of the subcloned B. subtilis EPSPS        (pMON21133) were determined as described above. The data were        analyzed graphically by the same methods used for the 1A2        isolate, and the results obtained were comparable to those        reported above for B. subtilis 1A2 culture.        Characterization of the EPSPS gene from Staphylococcus aureus

The kinetic properties of the S. aureus EPSPS expressed in E. coli weredetermined, including the specific activity, the appK_(m)(PEP), and theappK_(i)(glyphosate). The S. aureus EPSPS gene has been previouslydescribed (O'Connell et al., 1993)

The strategy taken for the cloning of the S. aureus EPSPS was polymerasechain reaction (PCR), utilizing the known nucleotide sequence of the S.aureus aroA gene encoding EPSPS (O'Cormell et al., 1993). The S. aureusculture (ATCC 35556) was fermeated in an M2 facility in three 250 mLshake flasks containing 55 mL of TYE (tryptone 5 g/L, yeast extract 3g/L, pH 6.8). The three flasks were inoculated with 1.5 mL each of asuspension made from freeze dried ATCC 35556 S. aureus cells in 90 mL ofPBS (phosphate-buffered saline) buffer. Flasks were incubated at 30° C.for 5 days while shaking at 250 rpm. The resulting cells were lysed(boiled in TE [tris/EDTA] buffer for 8 minutes) and the DNA utilized forPCR reactions. The EPSPS gene was amplified using PCR and engineeredinto an E. coli expression vector as follows:

-   -   (i) two oligonucleotides were synthesized which incorporated two        restriction enzyme recognition sites (NcoI and SacI) to the        sequences of the oligonucleotides:

(SEQ ID NO:47) GGGGCCATGGTAAATGAACAAATCATTG (SEQ ID NO:48)GGGGGAGCTCATTATCCCTCATTTTGTAAAAGC

-   -   (ii) The purified, PCR-amplified aroA gene from S. aureus was        digested using NcoI and SacI enzymes.    -   (iii) DNA of pMON 5723, which contains a pRecA bacterial        promoter and Gene10 leader sequence (Olins et al., 1988) was        digested NcoI and SacI and the 3.5 kb digestion product was        purified.    -   (iv) The S. aureus PCR product and the NcoI/SacI pMON 5723        fragment were ligated and transformed into E. coli JM101        competent cells.    -   (v) Two spectinomycin-resistant E. coli JM101 clones from above        (SA#2 and SA#3) were purified and transformed formed into a        competent aroA- E. coli strain, GB100

For complementation experiments SAGB#2 and SAGB#3 were utilized, whichcorrespond to SA#2 and SA#3, respectively, transformed into E. coliGB100. In addition, E. coli GB100 (negative control) and pMON 9563 (wtpetunia EPSPS, positive control) were tested for AroA complementation.The organisms were grown in minimal media plus and minus aromatic aminoacids. Later analyses showed that the SA#2 and SA#3 clones wereidentical, and they were assigned the plasmid identifier pMON21139.

SAGB#2 in E. coli GB100 (pMON21139) was also grown in M9 minimal mediaand induced with nalidixic acid. A negative control, E. coli GB100, wasgrown under identical conditions except the media was supplemented witharomatic amino acids. The cells were harvested, washed with 0.9% NaCl,and frozen at −80° C., for extraction and EPSPS analysis.

The frozen pMON21139 E. coli GB100 cell pellet from above was extractedand assayed for EPSPS activity as previously described. EPSPS assayswere performed using 1 mM phosphoenolpyruvate (PEP), 2 mMshikimate-3-phosphate (S3P), 0. 1 mM ammonium molybdate, 5 mM potassiumfluoride, pH 7.0, 25° C. The total assay volume was 50μL, whichcontained 10 μL of the undiluted desalted extract.

The results indicate that the two clones contain a functional aroA/EPSPSgene since they were able to grow in minimal media which contained noaromatic amino acids. As expected, the GB100 culture did not grow onminimal medium without aromatic amino acids (since no functional EPSPSis present), and the pMON9563 did confer growth in minimal media. Theseresults demonstrated the successful cloning of a functional EPSPS genefrom S. aureus. Both clones tested were identical, and the E. coliexpression vector was designated pMON21139.

The plasmid pMON21139 in E. coli GB100 was grown in M9 minimal media andwas induced with nalidixic acid to induce EPSPS expression driven fromthe RecA promoter. A desalted extract of the intracellular protein wasanalyzed for EPSPS activity, yielding an EPSPS specific activity of0.005 μmol/min mg. Under these assay conditions, the S. aureus EPSPSactivity was completely resistant to inhibition by 1 mM glyphosate.Previous analysis had shown that E. coli GB100 is devoid of EPSPSactivity.

The appK_(m)(PEP) of the S. aureus EPSPS was determined by measuring thereaction velocity of the enzyme (in crude bacterial extracts) at varyingconcentrations of PEP. The results were analyzed graphically usingseveral standard kinetic plotting methods. Data analysis using thehyperbolic, Lineweaver-Burke, and Eadie-Hofstee methods yieldedappK_(m)(PEP) constants of 7.5, 4.8, and 4.0 μM, respectively. Thesethree data treatments are in good agreement, and yield an average valuefor appK_(m)(PEP) of 5 μM.

Further information of the glyphosate tolerance of S. aureus EPSPS wasobtained by determining the reaction rates of the enzyme in the presenceof several concentrations of glyphosate, at a PEP concentration of 2 μM.These results were compared to the calculated maximal velocity of theEPSPS, and making the assumption that glyphosate is a competitiveinhibitor versus PEP for S. aureus EPSPS, as it is for all othercharacterized EPSPSs, an appK_(i)(glyphosate) was determinedgraphically. The appK_(i)(glyphosate) for S. aureus EPSPS estimatedusing this method was found to be 0.20 mM.

The EPSPS from S. aureus was found to be glyphosate-tolerant, with anappK_(i)(glyphosate) of approximately 0.2 mM. In addition, theappK_(m)(PEP) for the enzyme is approximately 5 μM, yielding aappK_(i)(glyphosate)/appK_(m)(PEP) of 40.

Alternative Isolation Protocols for Other Class II EPSPS StructuralGenes

A number of Class II genes have been isolated and described here. Whilethe cloning of the gene from CP4 was difficult due to the low degree ofsimilarity between the Class I and Class II enzymes and genes, theidentification of the other genes were greatly facilitated by the use ofthis first gene as a probe. In the cloning of the LBAA EPSPS gene, theCP4 gene probe allowed the rapid identification of cosmid clones and thelocalization of the intact gene to a small restriction fragment and someof the CP4 sequencing primers were also used to sequence the LBAA (andPG2982) EPSPS gene(s). The CP4 gene probe was also used to confirm thePG2982 gene clone. The high degree of similarity of the Class II EPSPSgenes may be used to identify and clone additional genes in much thesame way that Class I EPSPS gene probes have been used to clone otherClass I genes. An example of the latter was in the cloning of the A.thaliana EPSPS gene using the P. hybrida gene as a probe (Klee et al.,1987).

Glyphosate-tolerant EPSPS activity has been reported previously for EPSPsynthases from a number of sources. These enzymes have not beencharacterized to any extent in most cases. The use of Class I and ClassII EPSPS gene probes or antibody probes provide a rapid means ofinitially screening for the nature of the EPSPS and provide tools forthe rapid cloning and characterization of the genes for such enzymes.

Two of the three genes described were isolated from bacteria that wereisolated from a glyphosate treatment facility (Strains CP4 and LBAA).The third (PG2982) was from a bacterium that had been isolated from aculture collection strain. This latter isolation confirms that exposureto glyphosate is not a prerequisite for the isolation of highglyphosate-tolerant EPSPS enzymes and that the screening of collectionsof bacteria could yield additional isolates. It is possible to enrichfor glyphosate degrading or glyphosate resistant microbial populations(Quinn et al., 1988; Talbot et al., 1984) in cases where it was feltthat enrichment for such microorganisms would enhance the isolationfrequency of Class II EPSPS microorganisms. Additional bacteriacontaining class II EPSPS gene have also been identified. A bacteriumcalled C 12, isolated from the same treatment column beads as CP4 (seeabove) but in a medium in which glyphosate was supplied as both thecarbon and phosphorus source, was shown by Southern analysis tohybridize with a probe consisting of the CP4 EPSPS coding sequence. Thisresult, in conjunction with that for strain LBAA, suggests that thisenrichment method facilitates the identification of Class II EPSPSisolates. New bacterial isolates containing Class II EPSPS genes havealso been identified from environments other than glyphosate wastetreatment facilities. An inoculum was prepared by extracting soil (froma recently harvested soybean field in Jerseyville, Ill.) and apopulation of bacteria selected by growth at 28° C. in Dworkin-Fostermedium containing glyphosate at 10 mM as a source of carbon (and withcycloheximide at 100 μg/ml to prevent the growth of fungi). Upon platingon L-agar media, five colony types were identified. Chromosomal DNA wasprepared from 2ml L-broth cultures of these isolates and the presence ofa Class II EPSPS gene was probed using a the CP4 EPSPS coding sequenceprobe by Southern analysis under stringent hybridization and washingconditions. One of the soil isolates, S2, was positive by this screen.

Class II EPSPS enzymes are identifiable by an elevated Ki for glyphosateand thus the genes for these will impart a glyphosate tolerancephenotype in heterologous hosts. Expression of the gene from recombinantplasmids or phage may be achieved through the use of a variety ofexpression promoters and include the T7 promoter and polymerase. The T7promoter and polymerase system has been shown to work in a wide range ofbacterial (and mammalian) hosts and offers the advantage of expressionof many proteins that may be present on large cloned fragments.Tolerance to growth on glyphosate may be shown on minimal growth media.In some cases, other genes or conditions that may give glyphosatetolerance have been observed, including over expression ofbeta-lactamase, the igrA gene (Fitzgibbon and Braymer, 1990), or thegene for glyphosate oxidoreductase (PCT Pub. No. WO92/00377). These areeasily distinguished from Class II EPSPS by the absence of EPSPS enzymeactivity.

The EPSPS protein is expressed from the aroA gene (also called aroE insome genera, for example, in Bacillus) and mutants in this gene havebeen produced in a wide variety of bacteria. Determining the identity ofthe donor organism (bacterium) aids in the isolation of Class II EPSPSgene—such identification may be accomplished by standardmicro-biological methods and could include Gram stain reaction, growth,color of culture, and gas or acid production on different substrates,gas chromatography analysis of methylesters of the fatty acids in themembranes of the microorganism, and determination of the GC % of thegenome. The identity of the donor provides information that may be usedto more easily isolate the EPSPS gene. An AroA- host more closelyrelated to the donor organism could be employed to clone the EPSPS geneby complementation but this is not essential since complementation ofthe E. coli AroA mutant by the CP4 EPSPS gene was observed. In addition,the information on the GC content the genome may be used in choosingnucleotide probes—donor sources with high GC % would preferably use theCP4 EPSPS gene or sequences as probes and those donors with low GC wouldpreferably employ those from Bacillus subtilis, for example.Relationships between different EPSPS genes

The deduced amino acid sequences of a number of Class I and the Class IIEPSPS enzymes were compared using the Bestfit computer program providedin the UWGCG package (Devereux et al. 1984). The degree of similarityand identity as determined using this program is reported. The degree ofsimilarity/identity determined within Class I and Class II proteinsequences is remarkably high, for instance, comparing E. coli with S.typhimurium (similarity/identity=93%/88%) and even comparing E. coliwith a plant EPSPS (Petunia hybrida; 72%/55%). These data are shown inTable IV. The comparison of sequences between Class I and Class II,however, shows a much lower degree of relatedness between the Classes(similarity/identity=50-53%/23-30%). The display of the Bestfit analysisfor the E. coli (SEQ ID NO:8) and CP4 (SEQ ID NO:3) sequences shows thepositions of the conserved residues and is presented in FIG. 6. Previousanalyses of EPSPS sequences had noted the high degree of conservation ofsequences of the enzymes and the almost invariance of sequences in tworegions—the “20-35” and “95-107” regions (Gasser et al., 1988; numberedaccording to the Petunia EPSPS sequence)—and these regions are lessconserved in the case of CP4 and LBAA when compared to Class I bacterialand plant EPSPS sequences (see FIG. 6 for a comparison of the E. coliand CP4 EPSPS sequences with the E. coli sequence appearing as the topsequence in the Figure). The corresponding sequences in the CP4 Class IIEPSPS are:

PGDKSTSHRSFMGGL (SEQ ID NO:32) and LDFGNAATGCRLT. (SEQ ID NO:33)

These comparisons show that the overall relatedness of Class I and ClassII is EPSPS proteins is low and that sequences in putative conservedregions have also diverged considerably.

In the CP4 EPSPS an alanine residue is present at the “glycine101”position. The replacement of the conserved glycine (from the “95-107”region) by an alanine results in an elevated K_(i) for glyphosate and inan elevation in the K_(m) for PEP in Class I EPSPS. In the case of theCP4 EPSPS, which contains an alanine at this position, the K_(m) for PEPis in the low range, indicating that the Class II enzymes differ in manyaspects from the EPSPS enzymes heretofore characterized.

Within the Class II isolates, the degree of similarity/identity is ashigh as that noted for that within Class I (Table IVA). FIG. 7 displaysthe Bestfit computer program alignment of the CP4 (SEQ ID NO:3) and LBAA(SEQ ID NO:5) EPSPS deduced amino acid sequences with the CP4 sequenceappearing as the top sequence in the Figure. The symbols used in FIGS. 6and 7 are the standard symbols used in the Bestfit computer program todesignate degrees of similarity and identity.

TABLE IVA^(1.2) Comparison of relatedness of EPSPS protein sequencesComparison between Class I and Class II EPSPS protein sequencessimilarity identity S. cerevisiae vs. CP4 54 30 A. nidulans vs. CP4 5025 B. napus vs. CP4 47 22 A. thaliana vs. CP4 48 22 N. tabacum vs. CP450 24 L. esculentum vs. CP4 50 24 P. hybrida vs. CP4 50 23 Z. mays vs.CP4 48 24 S. gallinarum vs. CP4 51 25 S. typhimurium vs. CP4 51 25 S.typhi vs. CP4 51 25 K. pneumoniae vs. CP4 56 28 Y. enterocolitica vs.CP4 53 25 H. influenzae vs. CP4 53 27 P. multocida vs. CP4 55 30 A.salmonicida vs. CP4 53 23 B. pertussis vs. CP4 53 27 E. coli vs. CP4 5226 E. coli vs. LBAA 52 26 E. coli vs. B. subtilis 55 29 E. coli vs. D.nodosus 55 32 E. coli vs. S. aureus 55 29 E. coli vs. Synechocystis sp.PCC6803 53 30 Comparison between Class I EPSPS protein sequencessimilarity identity E. coli vs. S. typhimurium 93 88 P. hybrids vs. E.coli 72 55 Comparison between Class II EPSPS protein sequencessimilarity identity D. nodosus vs. CP4 62 43 LBAA vs. CP4 90 83 PG2892vs. CP4 90 83 S. aureus vs. CP4 58 34 B. subtills vs. CP4 59 41Synechocystis sp. PCC6803 vs. CP4 62 45 ¹The EPSPS sequences comparedhere were obtained from the following references: E. coli, Rogers etal., 1983; S. typhimurium, Stalker et al., 1985; Petunia hybrids, Shahet al., 1986; B. pertussis, Maskell et al., 1988; S. cerevisiae, Duncanet al., 1987, Synechocystis sp. PCC6803, Dalla Chiesa et al., 1994 andD. nodosus, Alm et al., 1994. ²“GAP” Program, Genetics Computer Group,(1991), Program Manual for the GCG Package, Version 7, April 1991, 575Science Drive, Madison, Wisconsin, USA 53711

The relative locations of the major conserved sequences among Class IIEPSP sythase which distinguishes this group from the Class I EPSPsynthases is listed below in Table IVB.

TABLE IVB Location of Conserved Sequences in Class II EPSP SynthasesSource Seq. 1¹ Seq. 2² Seq. 3³ Seq. 4⁴ CP4 start 200 26 173 271 end 20429 177 274 LBAA start 200 26 173 271 end 204 29 177 274 PG2982 start 20026 173 273 end 204 29 177 276 B. subtilis start 190 17 164 257 end 19420 168 260 S. aureus start 193 21 166 261 end 197 24 170 264Synechocystis sp. PCC6803 start 210 34 183 278 end 214 38 187 281 D.nodosus start 195 22 168 261 end 199 25 172 264 min. start 190 17 164257 max. end 214 38 187 281 ¹-R-X₁-H-X₂-E-(SEQ ID NO:37) ²-G-D-K-X₃-(SEQID NO:38) ³-S-A-Q-X₄-K-(SEQ ID NO:39) ⁴-N-X₅-T-E-(SEQ ID NO:40)

The domains of EPSP synthase sequence identified in this applicationwere determined to be those important for maintenance of glyphosateresistance and productive binding of PEP. The information used inindentifying these domains included sequence alignments of numerousglyphosate-sensitive EPSPS molecules and the three-dimensional x-raystructures of E. coli EPSPS (Stallings, et al. 1991) and CP4 EPSPS. Thestructures are representative of a glyphosate-sensitive (i.e., Class I)enzyme, and a naturally-occuring glyphosate-tolerant (i.e., Class II)enzyme of the present invention. These exemplary molecules weresuperposed three-dimensionally and the results displayed on a computergraphics terminal. Inspection of the display allowed for structure-basedfine-tuning of the sequence alignments of glyphosate-sensitive andglyphosate-resistant EPSPS molecules. The new sequence alignments wereexamined to determine differences between Class I and Class II EPSPSenzymes. Seven regions were identified and these regions were located inthe x-ray structure of CP4 EPSPS which also contained a bound analog ofthe intermediate which forms catalytically between PEP and S3P.

The structure of the CP4 EPSPS with the bound intermediate analog wasdisplayed on a computer graphics terminal and the seven sequencesegments were examined. Important residues for glyphosate binding wereidentified as well as those residues which stabilized the conformationsof those important residues; adjoining residues were considerednecessary for maintenance of correct three-dimensional structural motifsin the context of glyphosate- sensitive EPSPS molecules. Three of theseven domains were determined not to be important for glyphosatetolerance and maintenance of productive PEP binding. The following fourprimary domains were determined to be characteristic of Class II EPSPSenzymes of the present invention:

-   -   -R-XrH-X₂-E(SEQ ID NO:37), in which        -   X₁ is an uncharged polar or acidic amino acid,        -   X₂ is serine or threonine,        -   The Arginine (R) reside at position 1 is important because            the positive charge of its guanidium group destabilizes the            binding of glyphosate. The Histidine (H) residue at position            3 stabilizes the Arginine (R) residue at position 4 of SEQ            ID NO:40. The Glutamic Acid (E) residue at position 5            stabilizes the Lysine (K) residue at position 5 of SEQ ID            NO:39.    -   -G-D-K-X₃(SEQ ID NO:38), in which        -   X₃ is serine or threonine,    -    The Aspartic acid (D) residue at position 2 stabilizes the        Arginine (R) residue at position 4 of SEQ ID NO:40. The        Lysine (K) residue at position 3 is important because for        productive PEP binding.    -   -S-A-Q-X₄-K(SEQ ID NO:39), in which        -   X₄ is any amino acid,    -    The Alanine (A) residue at position 2 stabilizes the        Arginine (R) residue at position 1 of SEQ ID NO:37. The        Serine (S) residue at position 1 and the Glutamine (Q) residue        at position 3 are important for productive S3P binding.    -   -N-X₅-T-R(SEQ ID NO:40), in which        -   X₅ is any amino acid,    -    The Asparagine (N) residue at position 1 and the Threonine (T)        residue at position 3 stabilize residue X₁ at position 2 of SEQ        ID NO:37. The Arginine (R) residue at position 4 is important        because the positive charge of its guanidium group destabilizes        the binding of glyphosate.

Since the above sequences are only representative of the Class II EPSPSswhich would be included within the generic structure of this group ofEPSP synthases, the above sequences may be found within a subject EPSPsynthase molecule within slightly more expanded regions. It is believedthat the above-described conserved sequences would likely be found inthe following regions of the mature EPSP synthases molecule:

-   -   -R-X₁-H-X₂-E-(SEQ ID NO:37) located between amino acids 175 and        230 of the mature EPSP synthase sequence;    -   -G-D-K-X₃-(SEQ ID NO:38) located between amino acids 5 and 55 of        the mature EPSP synthase sequence;    -   -S-A-Q-X₄-K-(SEQ ID NO:39) located between amino acids 150 and        200 of the mature EPSP synthase sequence; and    -   -N-X₅-T-R-(SEQ ID NO:40) located between amino acids 245 and 295        of the mature EPSPS synthase sequence.

One difference that may be noted between the deduced amino acidsequences of the CP4 and LBAA EPSPS proteins is at position 100 where anAlanine is found in the case of the CP4 enzyme and a Glycine is found inthe case of the LBAA enzyme. In the Class I EPSPS enzymes a Glycine isusually found in the equivalent position, i.e Glycine96 in E. coli andK. pneumoniae and Glycine101 in Petunia. In the case of these threeenzymes it has been reported that converting that Glycine to an Alanineresults in an elevation of the appKi for glyphosate and a concomitantelevation in the appKm for PEP (Kishore et al., 1986; Kishore and Shah,1988; Sost and Amrhein, 1990), which, as discussed above, makes theenzyme less efficient especially under conditions of lower PEPconcentrations. The Glycine100 of the LBAA EPSPS was converted to anAlanine and both the appKm for PEP and the appKi for glyphosate weredetermined for the variant. The Glycine100Alanine change was introducedby mutagenesis using the following primer:

CGGCAATGCCGCCACCGGCGCGCGCC (SEQ ID NO:34)and both the wild type and variant genes were expressed in E. coli in aRecA promoter expression vector (pMON17201 and pMON17264, respectively)and the appKm's and appKi's determined in crude lysates. The dataindicate that the appKi(glyphosate) for the G100A variant is elevatedabout 16-fold (Table V). This result is in agreement with theobservation of the importance of this G-A change in raising theappKi(glyphosate) in the Class I EPSPS enzymes. However, in contrast tothe results in the Class I G-A variants, the appKm(PEP) in the Class II(LBAA) G-A variant is unaltered. This provides yet another distinctionbetween the Class II and Class I EPSPS enzymes.

TABLE V sppKi sppKm(PEP) (glyphosate) Lysato prepared from: E.coli/pMON17201 (wild 5.3 μM 28 μM* type) E. coli/pMON17264 5.5 μM 459μM# (G100A variant) @range of PEP; 2-40 μM *range of glyphosate; 0-310μM; #range of glyphosate; 0-5000 μM.The LBAA G100A variant, by virtue of its superior kinetic properties,should be capable of imparting improved in planta glyphosate tolerance.Modification and Resynthesis of the Agrobacterium sp. strain CP4 EPSPSGene Sequence

The EPSPS gene from Agrobacterium sp. strain CP4 contains sequences thatcould be inimical to high expression of the gene in plants. Thesesequences include potential polyadenylation sites that are often and A+Trich, a higher G+C % than that frequently found in plant genes (63%versus ˜50%), concentrated stretches of G and C residues, and codonsthat are not used frequently in plant genes. The high G+C % in the CP4EPSPS gene has a number of potential consequences including thefollowing: a higher usage of G or C than that found in plant genes inthe third position in codons, and the potential to form strong hair-pinstructures that may affect expression or stability of the RNA. Thereduction in the G+C content of the CP4 EPSPS gene, the disruption ofstretches of G's and C's, the elimination of potential polyadenylationsequences, and improvements in the codon usage to that used morefrequently in plant genes, could result in higher expression of the CP4EPSPS gene in plants.

A synthetic CP4 gene was designed to change as completely as possiblethose inimical sequences discussed above. In summary, the gene sequencewas redesigned to eliminate as much as possible the following sequencesor sequence features (while avoiding the introduction of unnecessaryrestriction sites): stretches of G's and C's of 5 or greater; and A+Trich regions (predominantly) that could function as polyadenylationsites or potential RNA destabilization region. The sequence of this geneis shown in FIG. 8 (SEQ ID NO:9). This coding sequence was expressed inE. coli from the RecA promoter and assayed for EPSPS activity andcompared with that from the native CP4 EPSPS gene. The apparent Km forPEP for the native and synthetic genes was 11.8 and 12.7, respectively,indicating that the enzyme expressed from the synthetic gene wasunaltered. The N-terminus of the coding sequence was mutagenized toplace an SphI site at the ATG to permit the construction of the CTP2-CP4synthetic fusion for chloroplast import. The following primer was usedto accomplish this mutagenesis:

(SEQ ID NO:35) GGACCGCTGCTTGCACCGTGAAGCATGCTTAAGCTTGGCGTAATCATGG.Expression of Chloroplast Directed CP4 EPSPS

The glyphosate target in plants, the 5-enolpyruvyl-shikimate-3-phosphatesynthase (EPSPS) enzyme, is located in the chloroplast. Manychloroplast-localized proteins, including EPSPS, are expressed fromnuclear genes as precursors and are targeted to the chloroplast by achloroplast transit peptide (CTP) that is removed during the importsteps. Examples of other such chloroplast proteins include the smallsubunit (SSU) of Ribulose-1,5-bisphosphate carboxylase (RUBISCO),Ferredoxia, Ferredoxin oxidoreductase, the Light-harvesting-complexprotein I and protein II, and Thioredoxin F. It has been demonstrated invivo and in vitro that non-chloroplast proteins may be targeted to thechloroplast by use of protein fusions with a CTP and that a CTP sequenceis sufficient to target a protein to the chloroplast.

A CTP-CP4 EPSPS fusion was constructed between the Arabidopsis thalianaEPSPS CTP (Klee et al., 1987) and the CP4 EPSPS coding sequences. TheArabidopsis CTP was engineered by site-directed mutagenesis to place aSphI restriction site at the CTP processing site. This mutagenesisreplaced the Glu-Lys at this location with Cys-Met. The sequence of thisCTP, designated as CTP2 (SEQ ID NO:10), is shown in FIG. 9. TheN-terminus of the CP4 EPSPS gene was modified to place a SphI site thatspans the Met codon. The second codon was converted to one for leucinein this step also. This change had no apparent effect on the in vivoactivity of CP4 EPSPS in E. coli as judged by rate of complementation ofthe aroA allele. This modified N-terminus was then combined with theSacI C-terminus and cloned downstream of the CTP2 sequences. TheCTP2-CP4 EPSPS fusion was cloned into pBlueScript KS(+). This vector maybe transcribed in vitro using the T7 polymerase and the RNA translatedwith ³⁵S-Methionine to provide material that may be evaluated for importinto chloroplasts isolated from Lactuca sativa using the methodsdescribed hereinafter (della-Cioppa et al., 1986, 1987). This templatewas transcribed in vitro using T7 polymerase and the³⁵S-methionine-labeled CTP2-CP4 EPSPS material was shown to import intochloroplasts with an efficiency comparable to that for the controlPetunia EPSPS (control=³⁵S labeled PreEPSPS [pMON6140; della-Cioppa etal., 1986]).

In another example the Arabidopsis EPSPS CTP, designated as CTP3, wasfused to the CP4 EPSPS through an EcoRI site. The sequence of this CTP3(SEQ ID NO:12) is shown in FIG. 10. An EcoRI site was introduced intothe Arabidopsis EPSPS mature region around amino acid 27, replacing thesequence -Arg-Ala-Leu-Leu- with -Arg-Ile-Leu-Leu- in the process. Theprimer of the following sequence was used to modify the N-terminus ofthe CP4 EPSPS gene to add an EcoRI site to effect the fusion to the

CTG3: GGAAGACGCCCAGATTCACGGTGCAAGCAGCCGG (the EcoRI site is underlined)(SEQ ID NO:36)This CTP3-CP4 EPSPS fusion was also cloned into the pBlueScript vectorand the T7 expressed fusion was found to also import into chloroplastswith an efficiency comparable to that for the control Petunia EPSPS(pMON6140).

A related series of CTPs, designated as CTP4 (SphI) and CTP5 (EcoRI),based on the Petunia EPSPS CTP and gene were also fused to the SphI- andEcoRI-modified CP4 EPSPS gene sequences. The SphI site was added bysite-directed mutagenesis to place this restriction site (and change theamino acid sequence to -Cys-Met-) at the chloroplast processing site.All of the CTP-CP4 EPSPS fusions were shown to import into chloroplastswith approximately equal efficiency. The CTP4 (SEQ ID NO:14) and CTP5(SEQ ID NO:16) sequences are shown in FIGS. 11 and 12.

A CTP2-LBAA EPSPS fusion was also constructed following the modificationof the N-terminus of the LBAA EPSPS gene by the addition of a SphI site.This fusion was also found to be imported efficiently into chloroplasts.

By similar approaches, the CTP2-CP4 EPSPS and the CTP4-CP4 EPSPS fusionhave also been shown to import efficiently into chloroplasts preparedfrom the leaf sheaths of corn. These results indicate that these CTP-CP4fusions could also provide useful genes to impart glyphosate tolerancein monocot species.

The use of CTP2 or CTP4 is preferred because these transit peptideconstructions yield mature EPSPS enzymes upon import into thechloroplast which are closer in composition to the native EPSPSs notcontaining a transit peptide signal. Those skilled in the art willrecognize that various chimeric constructs can be made which utilize thefunctionality of a particular CTP to import a Class II EPSPS enzyme intothe plant cell chloroplast. The chloroplast import of the Class II EPSPScan be determined using the following assay.

Chloroplast Uptake Assay

Intact chloroplasts are isolated from lettuce (Latuca sativa, var.longifolia) by centrifugation in Percoll/ficoll gradients as modifiedfrom Bartlett et al., (1982). The final pellet of intact chloroplasts issuspended in 0.5 ml of sterile 330 mM sorbitol in 50 mM Hepes-KOH, pH7.7, assayed for chlorophyll (Arnon, 1949), and adjusted to the finalchlorophyll concentration of 4 mg/ml (using sorbitol/Hepes). The yieldof intact chloroplasts from a single head of lettuce is 3-6 mgchlorophyll.

A typical 300 μl uptake experiment contained 5 mM ATP, 8.3 mM unlabeledmethionine, 322 mM sorbitol, 58.3 mM Hepes-KOH (pH 8.0), 50 μlreticulocyte lysate translation products, and intact chloroplasts fromL. sativa (200 μg chlorophyll). The uptake mixture is gently rocked atroom temperature (in 10×75 mm glass tubes) directly in front of a fiberoptic illuminator set at maximum light intensity (150 Watt bulb).Aliquot samples of the uptake mix (about 50 μl) are removed at varioustimes and fractionated over 100 μl silicone-oil gradients (in 150 μlpolyethylene tubes) by centrifugation at 11,000× g for 30 seconds. Underthese conditions, the intact chloroplasts form a pellet under thesilicone-oil layer and the incubation medium (containing thereticulocyte lysate) floats on the surface. After centrifugation, thesilicone-oil gradients are immediately frozen in dry ice. Thechloroplast pellet is then resuspended in 50-100 μl of lysis buffer (10mM Hepes-KOH pH 7.5, 1 mM PMSF, 1 mM benzamidine, 5 mM e-amino-n-caproicacid, and 30 μg/ml aprotinin) and centrifuged at 15,000× g for 20minutes to pellet the thylakoid membranes. The clear supernatant(stromal proteins) from this spin, and an aliquot of the reticulocytelysate incubation medium from each uptake experiment, are mixed with anequal volume of 2×SDS-PAGE sample buffer for electrophoresis (Laemmli,1970).

SDS-PAGE is carried out according to Laemmli (1970) in 3-17% (w/v)acrylamide slab gels (60 mm×1.5 mm) with 3% (w/v) acrylamide stackinggels (5 mm×1.5 mm). The gel is fixed for 20-30 rain in a solution with40% methanol and 10% acetic acid. Then, the gel is soaked in EN³HANCE™(DuPont) for 20-30 minutes, followed by drying the gel on a gel dryer.The gel is imaged by autoradiography, using an intensifying screen andan overnight exposure to determine whether the CP4 EPSPS is importedinto the isolated chloroplasts.

Plant Transformation

Plants which can be made glyphosate-tolerant by practice of the presentinvention include, but are not limited to, soybean, cotton, corn,canola, oil seed rape, flax, sugarbeet, sunflower, potato, tobacco,tomato, wheat, rice, alfalfa and lettuce as well as various tree, nutand vine species.

A double-stranded DNA molecule of the present invention (“chimericgene”) can be inserted into the genome of a plant by any suitablemethod. Suitable plant transformation vectors include those derived froma Ti plasmid of Agrobacterium tumefaciens, as well as those disclosed,e.g., by Herrera-Estrella (1983), Beyart (1984), Klee (1985) and EPOpublication 120,516 (Schilperoort et al.). In addition to planttransformation vectors derived from the Ti or root-inducing (Ri)plasmids of Agrobacterium, alternative methods can be used to insert theDNA constructs of this invention into plant cells. Such methods mayinvolve, for example, the use of liposomes, electroporation, chemicalsthat increase free DNA uptake, free DNA delivery via microprojectilebombardment, and transformation using viruses or pollen.

Class II EPSPS Plant transformation vectors

Class II EPSPS DNA sequences may be engineered into vectors capable oftransforming plants by using known techniques. The following descriptionis meant to be illustrative and not to be read in a limiting sense. Oneof ordinary skill in the art would know that other plasmids, vectors,markers, promoters, etc. would be used with suitable results. TheCTP2-CP4 EPSPS fusion was cloned as a BglII-EcoRI fragment into theplant vector pMON979 (described below) to form pMON17110, a map of whichis presented in FIG. 13. In this vector the CP4 gene is expressed fromthe enhanced CaMV35S promoter (E35S; Kay et al. 1987). A FMV35S promoterconstruct (pMON17116) was completed in the following way: The SaII-NotIand the NotI-BglII fragments from pMON979 containing theSpc/AAC(3)-III/oriV and the pBR322/Right Border/NOS 3′/CP4 EPSPS genesegment from pMON17110 were ligated with the XhoI-BglII FMV35S promoterfragment from pMON981. These vectors were introduced into tobacco,cotton and canola.

A series of vectors was also completed in the vector pMON977 in whichthe CP4 EPSPS gene, the CTP2-CP4 EPSPS fusion, and the CTP3-CP4 fusionwere cloned as BglII-SacI fragments to form pMON17124, pMON17119, andpMON17120, respectively. These plasmids were introduced into tobacco. ApMON977 derivative containing the CTP2-LBAA EPSPS gene was alsocompleted (pMON17206) and introduced into tobacco.

The pMON979 plant transformation/expression vector was derived frompMON886 (described below) by replacing the neomycin phosphotransferasetypeII (KAN) gene in pMON886 with the 0.89 kb fragment containing thebacterial gentamicin-3-N-acetyltransferase type III (AAC(3)-III) gene(Hayford et al., 1988). The chimeric P-35S/AA(3)-III/NOS 3′ gene encodesgentamicin resistance which permits selection of transformed plantcells. pMON979 also contains a 0.95 kb expression cassette consisting ofthe enhanced CaMV 35S promoter (Kay et al., 1987), several uniquerestriction sites, and the NOS 3′ end (P-En-CaMV35SfNOS 3′). The rest ofthe pMON979 DNA segments are exactly the same as in pMON886.

Plasmid pMON886 is made up of the following segments of DNA. The firstis a 0.93 kb AvaI to engineered-EcoRV fragment isolated from transposonTn7 that encodes bacterial spectinomycin/streptomycin resistance(Spc/Str), which is a determinant for selection in E. coli andAgrobacterium tumefaciens. This is joined to the 1.61 kb segment of DNAencoding a chimeric kanamycin resistance which permits selection oftransformed plant cells. The chimeric gene (P-35S/KANfNOS 3′) consistsof the cauliflower mosaic virus (CaMV) 35S promoter, the neomycinphosphotransferase typeII (KAN) gene, and the 3′-nontranslated region ofthe nopaline synthase gene (NOS 3′) (Fraley et al., 1983). The nextsegment is the 0.75 kb oriV containing the origin of replication fromthe RK2 plasmid. It is joined to the 3.1 kb SalI to PvuI segment ofpBR322 (ori322) which provides the origin of replication for maintenancein E. coli and the bom site for the conjugational transfer into theAgrobacterium tumefaciens cells. The next segment is the 0.36 kb PvuI toBclI from pTiT37 that carries the nopaline-type T-DNA right border(Fraley et al., 1985).

The pMON977 vector is the same as pMON981 except for the presence of theP-En-CaMV35S promoter in place of the FMV35S promoter (see below).

The pMON981 plasmid contains the following DNA segments: the 0.93 kbfragment isolated from transposon Tn7 encoding bacterialspectinomycin/streptomycin resistance [Spc/Str; a determinant forselection in E. coli and Agrobacterium tumefaciens (Fling et al.,1985)]; the chimeric kanamycin resistance gene engineered for plantexpression to allow selection of the transformed tissue, consisting ofthe 0.35 kb cauliflower mosaic virus 35S promoter (P-35S) (Odell et al.,1985), the 0.83 kb neomycin phosphotransferase typeII gene (KAN), andthe 0.26 kb 3′-nontranslated region of the nopaline synthase gene (NOS3′) (Fraley et al., 1983); the 0.75 kb origin of replication from theRK2 plasmid (oriV) (Stalker et al., 1981); the 3.1 kb SalI to PvuIsegment of pBR322 which provides the origin of replication formaintenance in E. coli (ori-322) and the bom site for the conjugationaltransfer into the Agrobacterium tumefaciens cells, and the 0.36 kb PvuIto BclI fragment from the pTiT37 plasmid containing the nopaline-typeT-DNA right border region (Fraley et al., 1985). The expression cassetteconsists of the 0.6 kb 35S promoter from the figwort mosaic virus(P-FMV35S) (Gowda et al., 1989) and the 0.7 kb 3′ non-translated regionof the pea rbcS-E9 gene (E9 3′) (Coruzzi et al., 1984, and Morelli etal., 1985). The 0.6 kb SspI fragment containing the FMV35S promoter(FIG. 1) was engineered to place suitable cloning sites downstream ofthe transcriptional start site. The CTP2-CP4syn gene fusion wasintroduced into plant expression vectors (including pMON981, to formpMON17131; FIG. 14) and transformed into tobacco, canola, potato,tomato, sugarbeet, cotton, lettuce, cucumber, oil seed rape, poplar, andArabidopsis.

The plant vector containing the Class II EPSPS gene may be mobilizedinto any suitable Agrobacterium strain for transformation of the desiredplant species. The plant vector may be mobilized into an ABIAgrobacterium strain. A suitable ABI strain is the A208 Agrobacteriumtumefaciens carrying the disarmed Ti plasmid pTiC58 (pMP90RK) (Koncz andSchell, 1986). The Ti plasmid does not carry the T-DNA phytohormonegenes and the strain is therefore unable to cause the crown galldisease. Mating of the plant vector into ABI was done by the triparentalconjugation system using the helper plasmid pRK2013 (Ditta et al.,1980). When the plant tissue is incubated with the ABI::plant vectorconjugate, the vector is transferred to the plant cells by the virfunctions encoded by the disarmed pTiC58 plasmid. The vector opens atthe T-DNA right border region, and the entire plant vector sequence maybe inserted into the host plant chromosome. The pTiC58 Ti plasmid doesnot transfer to the plant cells but remains in the Agrobacterium. ClassII EPSPS free DNA vectors

Class II EPSPS genes may also be introduced into plants through directdelivery methods. A number of direct delivery vectors were completed forthe CP4 EPSPS gene. The vector pMON13640, a map of which is presented inFIG. 15, is described here. The plasmid vector is based on a pUC plasmid(Vieira and Messing, 1987) containing, in this case, the nptII gene(kanamycin resistance; KAN) from Tn903 to provide a selectable marker inE. coli. The CTP4-EPSPS gene fusion is expressed from the P-FMV35Spromoter and contains the NOS 3′ polyadenylation sequence fragment andfrom a second cassette consisting of the E35S promoter, the CTP4-CP4gene fusion and the NOS 3′ sequences. The scoreable GUS marker gene(Jefferson et al., 1987) is expressed from the mannopine synthasepromoter (P-MAS; Velten et al., 1984) and the soybean 7S storage proteingene 3′ sequences (Schuler et al., 1982). Similar plasmids could also bemade in which CTP-CP4 EPSPS fusions are expressed from the enhancedCaMV35S promoter or other plant promoters. Other vectors could be madethat are suitable for free DNA delivery into plants and such are withinthe skill of the art and contemplated to be within the scope of thisdisclosure.

Plastid transformation:

While transformation of the nuclear genome of plants is much moredeveloped at this time, a rapidly advancing alternative is thetransformation of plant organelles. The transformation of plastids ofland plants and the regeneration of stable transformants has beendemonstrated (Svab et al., 1990; Maliga et al., 1993). Transformants areselected, following double cross-over events into the plastid genome, onthe basis of resistance to spectinomycin conferred through rRNA changesor through the introduction of an aminoglycoside 3″-adenyltransferasegene (Svab et al., 1990; Svab and Maliga, 1993), or resistance tokanamycin through the neomycin phosphotransferase NptII (Carrer et al.,1993). DNA is introduced by biolistic means (Svab et al, 1990; Maliga etal., 1993) or by using polyethylene glycol (O'Neill et al., 1993). Thistransformation route results in the production of 500-10,000 copies ofthe introduced sequence per cell and high levels of expression of theintroduced gene have been reported (Carrer et al., 1993; Maliga et al.,1993). The use of plastid transformation offers the advantages of notrequiring the chloroplast transit peptide signal sequence to result inthe localization of the heterologous Class II EPSPS in the chloroplastand the potential to have many copies of the heterologousplant-expressible Class II EPSPS gene in each plant cell since at leastone copy of the gene would be in each plastid of the cell.

Plant Regeneration

When expression of the Class II EPSPS gene is achieved in transformedcells (or protoplasts), the cells (or protoplasts) are regenerated intowhole plants. Choice of methodology for the generation step is notcritical, with suitable protocols being available for hosts fromLeguminosae (alfalfa, soybean, clover, etc.), Umbelliferae (carrot,celery, parsnip), Cruciferae (cabbage, radish, rapeseed, etc.),Cucurbitaceae (melons and cucumber), Gramineae (wheat, rice, corn,etc.), Solanaceae (potato, tobacco, tomato, peppers), various floralcrops as well as various trees such as poplar or apple, nut crops orvine plants such as grapes. See, e.g., Ammirato, 1984; Shimamoto, 1989;Fromm, 1990; Vasil, 1990.

The following examples are provided to better elucidate the practice ofthe present invention and should not be intrepreted in any way to limitthe scope of the present invention. Those skilled in the art willrecognize that various modifications, truncations, etc. can be made tothe methods and genes described herein while not departing from thespirit and scope of the present invention.

In the examples that follow, EPSPS activity in plants is assayed by thefollowing method. Tissue samples were collected and immediately frozenin liquid nitrogen. One gram of young leaf tissue was frozen in a mortarwith liquid nitrogen and ground to a fine powder with a pestle. Thepowder was then transferred to a second mortar, extraction buffer wasadded (1 ml/gram), and the sample was ground for an additional 45seconds. The extraction buffer for canola consists of 100 mM Tris, 1 mMEDTA, 10% glycerol, 5 mM DTT, 1 mM BAM, 5 mM ascorbate, 1.0 mg/ml BSA,pH 7.5 (4° C.). The extraction buffer for tobacco consists of 100 mMTris, 10 mM EDTA, 35 mM KCl, 20% glycerol, 5 mM DTT, 1 mM BAM, 5 mMascorbate, 1.0 mg/ml BSA, pH 7.5 (4° C.). The mixture was transferred toa microfuge tube and centrifuged for 5 minutes. The resultingsupernatants were desalted on spin G-50 (Pharmacia) columns, previouslyequilibrated with extraction buffer (without BSA), in 0.25 ml aliquots.The desalted extracts were assayed for EPSP synthase activity byradioactive HPLC assay. Protein concentrations in samples weredetermined by the BioRad microprotein assay with BSA as the standard.

Protein concentrations were determined using the BioRad Microproteinmethod, BSA was used to generate a standard curve ranging from 2-24 μg.Either 800 μl of standard or diluted sample was mixed with 200 μl ofconcentrated BioRad Bradford reagent. The samples were vortexed and readat A(595) after ˜5 minutes and compared to the standard curve.

EPSPS enzyme assays contained HEPES (50 mM), shikimate-3-phosphate (2mM), NH₄ molybdate (0.1 mM) and KF (5 mM), with or without glyphosate(0.5 or 1.0 mM). The assay mix (30 μl) and plant extract (10 μl) werepreincubated for 1 minute at 25° C. and the reactions were initiated byadding ¹⁴C-PEP (1 mM). The reactions were quenched after 3 minutes with50 μl of 90% EtOH/0.1M HOAc, pH 4.5. The samples were spun at 6000 rpmand the resulting supernatants were analyzed for ¹⁴C-EPSP production byHPLC. Percent resistant EPSPS is calculated from the EPSPS activitieswith and without glyphosate.

The percent conversion of ¹⁴C labeled PEP to ¹⁴C EPSP was determined byHPLC radioassay using a C18 guard column (Brownlee) and an AX₁₀₀ HPLCcolumn (0.4×25 cm, Synchropak) with 0.28M isocratic potassium phosphateeluant, pH 6.5, at 1 ml/min. Initial velocities were calculated bymultiplying fractional turnover per unit time by the initialconcentration of the labeled substrate (1 mM). The assay was linear withtime up to ˜3 minutes and 30% turnover to EPSPS. Samples were dilutedwith 10 mM Tris, 10% glycerol, 10 mM DTT, pH 7.5 (4° C.) if necessary toobtain results within the linear range.

In these assays DL-dithiotheitol (DTT), benzamidine (BAM), and bovineserum albumin (BSA, essentially globulin free) were obtained from Sigma.Phosphoenolpyruvate (PEP) was from Boehringer Mannheim andphosphoenol[1-¹⁴C]pyruvate (28 mCi/mmol) was from Amersham.

EXAMPLES Example 1

Transformed tobacco plants have been generated with a number of theClass II EPSPS gene vectors containing the CP4 EPSPS DNA sequence asdescribed above with suitable expression of the EPSPS. These transformedplants exhibit glyphosate tolerance imparted by the Class II CP4 EPSPS.

Transformation of tobacco employs the tobacco leaf disc transformationprotocol which utilizes healthy leaf tissue about 1 month old. After a15-20 minutes surface sterilization with 10% Clorox plus a surfactant,the leaves are rinsed 3 times in sterile water. Using a sterile paperpunch, leaf discs are punched and placed upside down on MS104 media (MSsalts 4.3 g/l, sucrose 30 g/l, B5vitamins 500×2 ml/l, NAA 0.1 mg/l, andBA 1.0 mg/l) for a 1 day preculture.

The discs are then inoculated with an overnight culture of a disarmedAgrobacterium ABI strain containing the subject vector that had beendiluted 1/5 (i.e.: about 0.6 OD). The inoculation is done by placing thediscs in centrifuge tubes with the culture. After 30 to 60 seconds, theliquid is drained off and the discs were blotted between sterile filterpaper. The discs are then placed upside down on MS104 feeder plates witha filter disc to co-culture.

After 2-3 days of co-culture, the discs are transferred, still upsidedown, to selection plates with MS104 media. After 2-3 weeks, callustissue formed, and individual clumps are separated from the leaf discs.Shoots are cleanly cut from the callus when they are large enough to bedistinguished from stems. The shoots are placed on hormone-free rootingmedia (MSO: MS salts 4.3 g/l, sucrose 30 g/l, and B5 vitamins 500×2ml/l) with selection for the appropriate antibiotic resistance. Rootformation occurred in 1-2 weeks. Any leaf callus assays are preferablydone on rooted shoots while still sterile. Rooted shoots are then placedin soil and kept in a high humidity environment (i.e.: plasticcontainers or bags). The shoots are hardened off by gradually exposingthem to ambient humidity conditions.

Expression of CP4 EPSPS protein in transformed plants

Tobacco cells were transformed with a number of plant vectors containingthe native CP4 EPSPS gene, and using different promoters and/or CTP's.Preliminary evidence for expression of the gene was given by the abilityof the leaf tissue from antibiotic selected transformed shoots torecallus on glyphosate. In some cases, glyphosate-tolerant callus wasselected directly following transformation. The level of expression ofthe CP4 EPSPS was determined by the level of glyphosate-tolerant EPSPSactivity (assayed in the presence of 0.5 mM glyphosate) or by Westernblot analysis using a goat anti-CP4 EPSPS antibody. The Western blotswere quantitated by densitometer tracing and comparison to a standardcurve established using purified CP4 EPSPS. These data are presented as% soluble leaf protein. The data from a number of transformed plantlines and transformation vectors are presented in Table VI below.

TABLE VI Expression of CP4 EPSPS in transformed tobacco tissue CP4EPSPS** Vector Plant # (% leaf protein) pMON17110 25313 0.02 pMON1711025329 0.04 pMON17116 25095 0.02 pMON17119 25106 0.09 pMON17119 257620.09 pMON17119 25767 0.03 **Glyphosate-tolerant EPSPS activity was alsodemonstrated in leaf extracts for these plants.

Glyphosate tolerance has also been demonstrated at the whole plant levelin transformed tobacco plants. In tobacco, R_(o) transformants ofCTP2-CP4 EPSPS were sprayed at 0.4 lb/acre (0.448 kg/hectare), a ratesufficient to kill control non-transformed tobacco plants correspondingto a rating of 3, 1 and 0 at days 7, 14 and 28, respectively, and wereanalyzed vegetatively and reproductively (Table VII).

TABLE VII Glyphosate tolerance in R_(o) tobacco CP4 transformants*Score** Vegetative Vector/Plant # day 7 day 14 day 28 FertilepMON17110/25313 6 4 2 no pMON17110/25329 9 10 10 yes pMON17119/25106 9 910 yes *Spray rate = 0.4 lb/acre (0.448 kg/hectare) **Plants areevaluated on a numerical scoring system of 0-10 where a vegetative scoreof 10 represents no damage relative to nonsprayed controls and 0represents a dead plant. Reproductive scores (Fertile) are determined at28 days after spraying and are evaluated as to whether or not the plantis fertile.

Example 2A

Canola plants were transformed with the pMON17110, pMON17116, andpMON17131 vectors and a number of plant lines of the transformed canolawere obtained which exhibit glyphosate tolerance.

Plant Material

Seedlings of Brassica napus cv Westar were established in 2 inch (˜5 cm)pots containing Metro Mix 350. They were grown in a growth chabmer at24° C., 16/8 hour photoperiod, light intensity of 400 μEm⁻²sec⁻¹ (HIDlamps). They were fertilized with Peters 20-10-20 General PurposeSpecial. After 2½ weeks they were transplanted to 6 inch (˜15 cm) potsand grown in a growth chamber at 15°/10° C. day/night temperature, 16/8hour photoperiod, light intensity of 800 uEm⁻²sec⁻¹ (HID lamps). Theywere fertilized with Peters 15-30-15 Hi-Phos Special.

Transformation/Selection/Regeneration

Four terminal internodes from plants just prior to bolting or in theprocess of bolting but before flowering were removed and surfacedsterilized in 70% v/v ethanol for 1 minute, 2% w/v sodium hypochloritefor 20 minutes and rinsed 3 times with sterile deionized water. Stemswith leaves attached could be refrigerated in moist plastic bags for upto 72 hours prior to sterilization. Six to seven stem segments were cutinto 5 mm discs with a Redco Vegetable Slicer 200 maintainingorientation of basal end.

The Agrobacterium was grown overnight on a rotator at 24° C. in 2 mls ofLuria Broth containing 50 mg/l kanamycin, 24 mg/l chloramphenicol and100 mg/l spectinomycin. A 1:10 dilution was made in MS (Murashige andSkoog) media giving approximately 9×10⁸ cells per ml. This was confirmedwith optical density readings at 660 mu. The stem discs (explants) wereinoculated with 1.0 ml of Agrobacterium and the excess was aspiratedfrom the explants.

The explants were placed basal side down in petri plates containing1/10× standard MS salts, B5 vitamins, 3% sucrose, 0.8% agar, pH 5.7, 1.0mg/l 6-benzyladenine (BA). The plates were layered with 1.5 ml of mediacontaining MS salts, B5 vitamins, 3% sucrose, pH 5.7, 4.0 mg/lp-chlorophenoxyacetic acid, 0.005 mg/l kinetin and covered with sterilefilter paper.

Following a 2 to 3 day co-culture, the explants were transferred to deepdish petri plates containing MS salts, B5 vitamins, 3% sucrose, 0.8%agar, pH 5.7, 1 mg/l BA, 500 mg/l carbenicillin, 50 mg/l cefotaxime, 200mg/l kanamycin or 175 mg/l gentamicin for selection. Seven explants wereplaced on each plate. After 3 weeks they were transferred to freshmedia, 5 explants per plate. The explants were cultured in a growth roomat 25° C., continuous light (Cool White).

Expression Assay

After 3 weeks shoots were excised from the explants. Leaf recallusingassays were initiated to confirm modification of R_(o) shoots. Threetiny pieces of leaf tissue were placed on recallusing media containingMS salts, B5 vitamins, 3% sucrose, 0.8% agar, pH 5.7, 5.0 mg/l BA, 0.5mg/l naphthalene acetic acid (NAA), 500 mg/l carbenicillin, 50 mg/lcefotaxime and 200 mg/l kanamycin or gentamicin or 0.5 mM glyphosate.The leaf assays were incubated in a growth room under the sameconditions as explant culture. After 3 weeks the leaf recallusing assayswere scored for herbicide tolerance (callus or green leaf tissue) orsensitivity (bleaching).

Transplantation

At the time of excision, the shoot stems were dipped in Rootone® andplaced in 2 inch (˜5 cm) pots containing Metro-Mix 350 and placed in aclosed humid environment. They were placed in a growth chamber at 24°C., 16/8 hour photoperiod, 400 uEm⁻¹sec⁻²(HID lamps) for a hardening-offperiod of approximately 3 weeks.

The seed harvested from R_(o) plants is R₁ seed which gives rise to R₁plants. To evaluate the glyphosate tolerance of an R_(o) plant, itsprogeny are evaluated. Because an R_(o) plant is assumed to behemizygous at each insert location, selfing results in maximum genotypicsegregation in the R₁. Because each insert acts as a dominant allele, inthe absence of linkage and assuming only one hemizygous insert isrequired for tolerance expression, one insert would segregate 3:1, twoinserts, 15:1, three inserts 63:1, etc. Therefore, relatively few R₁plants need be grown to find at least one resistant phenotype.

Seed from an R_(o) plant is harvested, threshed, and dried beforeplanting in a glyphosate spray test. Various techniques have been usedto grow the plants for R₁ spray evaluations. Tests are conducted in bothgreenhouses and growth chambers. Two planting systems are used; ˜10 cmpots or plant trays containing 32 or 36 cells. Soil used for planting iseither Metro 350 plus three types of slow release fertilizer or plantMetro 350. Irrigation is either overhead in greenhouses orsub-irrigation in growth chambers. Fertilizer is applied as required inirrigation water. Temperature regimes appropriate for canola weremaintained. A sixteen hour photoperiod was maintained. At the onset offlowering, plants are transplanted to ˜15 cm pots for seed production.

A spray “batch” consists of several sets of R₁ progenies all sprayed onthe same date. Some batches may also include evaluations of other thanR₁ plants. Each batch also includes sprayed and unsprayed non-transgenicgenotypes representing the genotypes in the particular batch which wereputatively transformed. Also included in a batch is one or morenon-segregating transformed genotypes previously identified as havingsome resistance.

Two-six plants from each individual R_(o) progeny are not sprayed andserve as controls to compare and measure the glyphosate tolerance, aswell as to assess any variability not induced by the glyphosate. Whenthe other plants reach the 2-4 leaf stage, usually 10 to 20 days afterplanting, glyphosate is applied at rates varying from 0.28 to 1.12kg/ha, depending on objectives of the study. Low rate technology usinglow volumes has been adopted. A laboratory track sprayer has beencalibrated to deliver a rate equivalent to field conditions.

A scale of 0 to 10 is used to rate the sprayed plants for vegetativeresistance. The scale is relative to the unsprayed plants from the sameR_(o) plant. A 0 is death, while a 10 represents no visible differencefrom the unsprayed plant. A higher number between 0 and 10 representsprogressively less damage as compared to the unsprayed plant. Plants arescored at 7, 14, and 28 days after treatment (DAT), or until bolting,and a line is given the average score of the sprayed plants within anR_(o) plant family.

Six integers are used to qualitatively describe the degree ofreproductive damage from glyphosate:

0: No floral bud development

2: Floral buds present, but aborted prior to opening

4: Flowers open, but no anthers, or anthers fail to extrude past petals

6: Sterile anthers

8: Partially sterile anthers

10: Fully fertile flowers

Plants are scored using this scale at or shortly after initiation offlowering, depending on the rate of floral structure development.

Expression of EPSPS in Canola

After the 3 week period, the transformed canola plants were assayed forthe presence of glyphosate-tolerant EPSPS activity (assayed in thepresence of glyphosate at 0.5 mM). The results are shown in Table VIII.

TABLE VIII Expression of CP4 EPSPS in transformed Canola plants %resistant EPSPS activity of Leaf extract Plant # (at 0.5 mM glyphosate)Vector Control 0 pMON17110 41 47 pMON17110 52 28 pMON17110 71 82pMON17110 104 75 pMON17110 172 84 pMON17110 177 85 pMON17110 252 29*pMON17110 350 49 pMON17116 40 25 pMON17116 99 87 pMON17116 175 94pMON17116 178 43 pMON17116 182 18 pMON17116 252 69 pMON17116 298 44*pMON17116 332 89 pMON17116 383 97 pMON17116 395 52 *assayed in thepresence of 1.0 mM glyphosate

R₁ transformants of canola were then grown in a growth chamber andsprayed with glyphosate at 0.56 kg/ha (kilogram/hectare) and ratedvegetatively. These results are shown in Table IXA-IXC. It is to benoted that expression of glyphosate resistant EPSPS in all tissues ispreferred to observe optimal glyphosate tolerance phenotype in thesetransgenic plants. In the Tables below, only expression results obtainedwith leaf tissue are described.

TABLE IXA Glyphosate tolerance in Class II EPSPS canola R₁ transformants(pMON17110 = P-E35S; pMON17116 = P-FMV35S; R1 plants; Spray rate = 0.56kg/ha) Vegetative % resistant Score** Vector/Plant No. EPSPS* day 7 day14 Control Westar 0 5 3 pMON17110/41 47 6 7 pMON17110/71 82 6 7pMON17110/177 85 9 10 pMON17116/40 25 9 9 pMON17116/99 87 9 10pMON17116/175 94 9 10 pMON17116/178 43 6 3 pMON17116/182 18 9 10pMON17116/383 97 9 10

TABLE IXB Glyphosate tolerance in Class II EPSPS canola R₁ transformants(pMON17131 = P-FWV35S; R1 plants; Spray rate = 0.84 kg/ha) Vegetativescore** Reproductive score Vector/Plant No. day 14 day 28 17131/78 10 1017131/102 9 10 17131/115 9 10 17131/116 9 10 17131/157 9 10 17131/169 1010 17131/255 10 10 control Westar 1 0

TABLE IXC Glyphosate tolerance in Class I EPSPS canola transformants(P-E35S; R2 Plants; Spray rate = 0.28 kg/ha) Vegetative % resistantScore** Vector/Plant No. EPSPS* day 7 day 14 Control Westar 0 4 2pMON899/715 96 5 6 pMON899/744 95 8 8 pMON899/794 86 6 4 pMON899/818 817 8 pMON899/885 57 7 6 *% resistant EPSPS activity in the presence of0.5 mM glyphosate **A vegetative score of 10 indicates no damage, ascore of 0 is given to a dead plant.

The data obtained for the Class II EPSPS transformants may be comparedto glyphosate-tolerant Class I EPSP transformants in which the samepromoter is used to express the EPSPS genes and in which the level ofglyphosate-tolerant EPSPS activity was comparable for the two types oftransformants. A comparison of the data of pMON17110 [in Table IXA] andpMON17131 [Table IXB] with that for pMON899 [in Table IXC; the Class Igene in pMON899 is that from A. thaliana {Klee et al., 1987} in whichthe glycine at position 101 was changed to an alanine] illustrates thatthe Class II EPSPS is at least as good as that of the Class I EPSPS. Animprovement in vegetative tolerance of Class II EPSPS is apparent whenone takes into account that the Class II plants were sprayed at twicethe rate and were tested as R₁ plants.

Example 2B

The construction of two plant transformation vectors and thetransformation procedures used to produce glyphosate-tolerant canolaplants are described in this example The vectors, pMON17209 andpMON17237, were used to generate transgenic glyphosate-tolerant canolalines. The vectors each contain the gene encoding the5-enol-pyruvyl-shikimate-3-phosphate synthase (EPSPS) from Agrobacteriumsp. strain CP4. The vectors also contain either the gox gene encodingthe glyphosate oxidoreductase enzyme (GOX) from Achromobacter sp. strainLBAA (Barry et al., 1992) or the gene encoding a variant of GOX (GOXv.247) which displays improved catalytic properties. These enzymesconvert glyphosate to aminomethylphosphonic acid and glyoxylate andprotect the plant from damage by the metabolic inactivation ofglyphosate. The combined result of providing an alternative, resistantEPSPS enzyme and the metabolism of glyphosate produces transgenic plantswith enhanced tolerance to glyphosate

Molecular biology techniques. In general, standard molecular biology andmicrobial genetics approaches were employed (Maniatis et al., 1982).Site-directed mutageneses were carried out as described by Kunkel et al.(1987). Plant-preferred genes were synthesized and the sequenceconfirmed.

Plant transformation vectors. The following describes the generalfeatures of the plant transformation vectors that were modified to formvectors pMON17209 and pMON17237. The Agrobacterium mediated planttransformation vectors contain the following well-characterized DNAsegments which are required for replication and function of the plasmids(Rogers and Klee, 1987; Klee and Rogers, 1989). The first segment is the0.45 kb ClaI-DraI fragment from the pTi15955 octopine Ti plasmid whichcontains the T-DNA left border region (Barker et al., 1983). It isjoined to the 0.75 kb origin of replication (oriV) derived from thebroad-host range plasmid RK2 (Stalker et al., 1981). The next segment isthe 3.1 kb SalI-PvuI segment of pBR₃₂₂ which provides the origin ofreplication for maintenance in E. coli and the born site for theconjugational transfer into the Agrobacterium turnefaciens cells(Bolivar et al., 1977). This is fused to the 0.93 kb fragment isolatedfrom transposon Tn7 which encodes bacterial spectinomycin andstreptomycin resistance (Fling et al., 1985), a determinant for theselection of the plasmids in E. coli and Agrobacterium. It is fused tothe 0.36 kb PvuI-BclI fragment from the pTiT37 plasmid which containsthe nopaline-type T-DNA right border region (Fraley et al., 1985).Several chimeric genes engineered for plant expression can be introducedbetween the Ti right and left border regions of the vector. In additionto the elements described above, this vector also includes the 35Spromoter/NPTII/NOS 3′ cassette to enable selection of transformed planttissues on kanamycin (Klee and Rogers, 1989; Fraley et al., 1983; andOdell, et al., 1985) within the borders. An “empty” expression cassetteis also present between the borders and consists of the enhanced E35Spromoter (Kay et al., 1987), the 3′ region from the small subunit ofRUBP carboxylase of pea (E9) (Coruzzi et al., 1984; Morelli et al.,1986), and a number of restriction enzyme sites that may be used for thecloning of DNA sequences for expression in plants. The planttransformation system based on Agrobacterium tumefaciens delivery hasbeen reviewed (Klee and Rogers, 1989; Fraley et al., 1986). TheAgrobacterium mediated transfer and integration of the vector T-DNA intothe plant chromosome results in the expression of the chimeric genesconferring the desired phenotype in plants.

Bacterial Inoculum. The binary vectors are mobilized into Agrobacteriumtumefaciens strain ABI by the triparental conjugation system using thehelper plasmid pRK2013 (Ditta et al., 1980). The ABI strain contains thedisarmed pTiC58 plasmid pMP90RK (Koncz and Schell, 1986) in thechloramphenicol resistant derivative of the Agrobacterium tumefaciensstrain A208.

Transformation procedure. Agrobacterium inocula were grown overnight at28° C. in 2 ml of LBSCK (LBSCK is made as follows: LB liquid medium [1liter volume]=10 g NaCl; 5 g Yeast Extract; 10 g tryptone; pH 7.0, andautoclave for 22 minutes. After autoclaving, add spectinomycin (50 mg/mlstock)—2 ml, kanamycin (50 mg/ml stock)—1 ml, and chloramphenicol (25mg/ml stock)—1 ml.). One day prior to inoculation, the Agrobacterium wassubcultured by inoculating 200 μl into 2 ml of fresh LBSCK and grownovernight. For inoculation of plant material, the culture was dilutedwith MSO liquid medium to an A₆₆₀ range of 0.2-0.4.

Seedlings of Brassica napus cv. Westar were grown in Metro Mix 350(Huminert Seed Co., St. Louis, Mo.) in a growth chamber with a day/nighttemperature of 15°/10° C., relative humidity of 50%, 16h/8h photoperiod,and at a light intensity of 500 μmol m⁻² sec⁻¹. The plants were watereddaily (via sub-irrigation) and fertilized every other day with Peter's15:30:15 (Fogelsville, Pa.).

In general, all media recipes and the transformation protocol followthose in Fry et al. (1987). Five to six week-old Westar plants wereharvested when the plants had bolted (but prior to flowering), theleaves and buds were removed, and the 4-5 inches of stem below theflower buds were used as the explant tissue source. Followingsterilization with 70% ethanol for 1 min and 38% Clorox for 20 min, thestems were rinsed three times with sterile water and cut into 5 mm-longsegments (the orientation of the basal end of the stem segments wasnoted). The plant material was incubated for 5 minutes with the dilutedAgrobacterium culture at a rate of 5 ml of culture per 5 stems. Thesuspension of bacteria was removed by aspiration and the explants wereplaced basal side down—for an optimal shoot regeneration response—ontoco-culture plates ( 1/10 MSO solid medium with a 1.5 ml TXD (tobaccoxanthi diploid) liquid medium overlay and covered with a sterile 8.5 cmfilter paper). Fifty-to-sixty stem explants were placed onto eachco-culture plate.

After a 2 day co-culture period, stem explants were moved onto MS mediumcontaining 750 mg/l carbenicillin, 50 mg/l cefotaxime, and 1 mg/l BAP(benzylaminopurine) for 3 days. The stem explants were then placed fortwo periods of three weeks each, again basal side down and with 5explants per plate, onto an MS/0.1 mM glyphosate, selection medium (alsocontaining carbenicillin, cefotaxime, and BAP (The glyphosate stock[0.5M] is prepared as described in the following: 8.45 g glyphosate[analytical grade] is dissolved in 50 ml deionized water, adding KOHpellets to dissolve the glyphosate, and the volume is brought to 100 mlfollowing adjusting the pH to 5.7. The solution is filter-sterilized andstored at 4° C.). After 6 weeks on this glyphosate selection medium,green, normally developing shoots were excised from the stem explantsand were placed onto fresh MS medium containing 750 mg/l carbenicillin,50 mg/l cefotaxime, and 1 mg/l BAP, for further shoot development. Whenthe shoots were 2-3 inches tall, a fresh cut at the end of the stem wasmade, the cut end was dipped in Root-tone, and the shoot was placed inMetro Mix 350 soil and allowed to harden-off for 2-3 weeks.

Construction of Canola transformation vector pMON17209. The EPSPS genewas isolated originally from Agrobacterium sp. strain CP4 and expressesa highly tolerant enzyme. The original gene contains sequences thatcould be inimical to high expression of the gene in some plants. Thesesequences include potential polyadenylation sites that are often A+Trich, a higher G+C % than that frequently found in dicotyledonous plantgenes (63% versus ˜50%), concentrated stretches of G and C residues, andcodons that may not used frequently in dicotyledonous plant genes. Thehigh G+C % in the CP4 EPSPS gene could also result in the formation ofstrong hairpin structures that may affect expression or stability of theRNA. A plant preferred version of the gene was synthesized and used forthese vectors. This coding sequence was expressed in E. coli from aPRecA-gene10L vector (Olins et al., 1988) and the EPSPS activity wascompared with that from the native CP4 EPSPS gene. The appK_(m) for PEPfor the native and synthetic genes was 11.8 μM and 12.7 μM,respectively, indicating that the enzyme expressed from the syntheticgene was unaltered. The N-terminus of the coding sequence was thenmutagenized to place an SphI site (GCATGC) at the ATG to permit theconstruction of the CTP2-CP4 synthetic fusion for chloroplast import.This change had no apparent effect on the in vivo activity of CP4 EPSPSin E. coli as judged by complementation of the aroA mutant. A CTP-CP4EPSPS fusion was constructed between the Arabidopsis thaliana EPSPS CTP(Klee et al., 1987) and the CP4 EPSPS coding sequences. The ArabidopsisCTP was engineered by site-directed mutagenesis to place a SphIrestriction site at the CTP processing site. This mutagenesis replacedthe Glu-Lys at this location with Cys-Met. The CTP2-CP4 EPSPS fusion wastested for import into chloroplasts isolated from Lactuca sativa usingthe methods described previously (della-Cioppa et al., 1986; 1987).

The GOX gene that encodes the glyphosate metabolizing enzyme glyphosateoxidoreductase (GOX) was cloned originally from Achromobacter sp. strainLBAA (Hallas et al., 1988; Barry et al., 1992). The gox gene from strainLBAA was also resynthesized in a plant-preferred sequence version and inwhich many of the restriction sites were removed (PCT Appln. No. WO92/00377). The GOX protein is targeted to the plastids by a fusionbetween the C-terminus of a CTP and the N-terminus of GOX. A CTP,derived from the SSU1A gene from Arabidopsis thaliana (Timko et al.,1988) was used. This CTP (CTP1) was constructed by a combination ofsite-directed mutageneses. The CTP1 is made up of the SSU1A CTP (aminoacids 1-55), the first 23 amino acids of the mature SSU1A protein(56-78), a serine residue (amino acid 79), a new segment that repeatsamino acids 50 to 56 from the CTP and the first two from the matureprotein (amino acids 80-87), and an alanine and methionine residue(amino acid 88 and 89). An NcoI restriction site if located at the 3′end (spans the Met89 codon) to facilitate the construction of precisefusions to the 5′ of GOX. At a later stage, a BglII site was introducedupstream of the N-terminus of the SSU1A sequences to facilitate theintroduction of the fusions into plant transformation vectors. A fusionwas assembled between CTP1 and the synthetic GOX gene.

The CP4 EPSPS and GOX genes were combined to form pMON17209 as describedin the following. The CTP2-CP4 EPSPS fusion was assembled and insertedbetween the constitutive FMV35S promoter (Gowda et al., 1989; Richins etal., 1987) and the E9 3′ region (Coruzzi et al., 1984; Morelli et al.,1985) in a pUC vector (Yannisch-Perron et al., 1985; Vieira and Messing,1987) to form pMON17190; this completed element may then be moved easilyas a NotI-NotI fragment to other vectors. The CTP1-GOX fusion was alsoassembled in a pUC vector with the FMV35S promoter. This element wasthen moved as a HindIII-BamHI fragment into the plant transformationvector pMON10098 and joined to the E9 3′ region in the process. Theresultant vector pMON17193 has a single NotI site into which the FMV35S/CTP2-CP4 EPSPS/E9 3′ element from pMON17190 was cloned to formpMON17194. The kanamycin plant transformation selection cassette (Fraleyet al., 1985) was then deleted from pMON17194, by cutting with XhoI andre-ligating, to form the pMON17209 vector (FIG. 24).

Construction of Canola transformation vector pMON17237. The GOX enzymehas an apparent Km for glyphosate [appK_(m)(glyphosate)] of ˜25 mM. Inan effort to improve the effectiveness of the glyphosate metabolic ratein plants, a variant of GOX has been identified in which theappK_(m)(glyphosate) has been reduced approximately 10-fold; thisvariant is referred to as GOX v.247 and the sequence differences betweenit and the original plant-preferred GOX are illustrated in PCT Appln.No. WO 92/00377. The GOX v.247 coding sequence was combined with CTP1and assembled with the FMV35S promoter and the E9 3′ by cloning into thepMON17227 plant transformation vector to form pMON17241. In this vector,effectively, the CP4 EPSPS was replaced by GOX v.247. The pMON17227vector had been constructed by replacing the CTP1-GOX sequence inpMON17193 with those for the CTP2-CP4 EPSPS, to form pMON17199 andfollowed by deleting the kanamycin cassette (as described above forpMON17209). The pMON17237 vector (FIG. 25) was then completed by cloningthe FMV35S/CTP2-CP4 EPSPS/E9 3′ element as a NotI-NotI fragment intopMON17241.

Example 3

Soybean plants were transformed with the pMON13640 (FIG. 15) vector anda number of plant lines of the transformed soybean were obtained whichexhibit glyphosate tolerance.

Soybean plants are transformed with pMON13640 by the method ofmicroprojectile injection using particle gun technology as described inChristou et al. (1988). The seed harvested from R_(o) plants is R₁ seedwhich gives rise to R₁ plants. To evaluate the glyphosate tolerance ofan R_(o) plant, its progeny are evaluated. Because an R_(o) plant isassumed to be hemizygous at each insert location, selfing results inmaximum genotypic segregation in the R₁. Because each insert acts as adominant allele, in the absence of linkage and assuming only onehemizygous insert is required for tolerance expression, one insert wouldsegregate 3:1, two inserts, 15:1, three inserts 63:1, etc. Therefore,relatively few R₁ plants need be grown to find at least one resistantphenotype.

Seed from an R_(o) soybean plant is harvested, and dried before plantingin a glyphosate spray test. Seeds are planted into 4 inch (˜5 cm) squarepots containing Metro 350. Twenty seedlings from each R_(o) plant isconsidered adequate for testing. Plants are maintained and grown in agreenhouse environment. A 12.5-14 hour photoperiod and temperatures of30° C. day and 24° C. night is regulated. Water soluble Peters Pete Litefertilizer is applied as needed.

A spray “batch” consists of several sets of R₁ progenies all sprayed onthe same date. Some batches may also include evaluations of other thanR₁ plants. Each batch also includes sprayed and unsprayed non-transgenicgenotypes representing the genotypes in the particular batch which wereputatively transformed. Also included in a batch is one or morenon-segregating transformed genotypes previously identified as havingsome resistance.

One to two plants from each individual R_(o) progeny are not sprayed andserve as controls to compare and measure the glyphosate tolerance, aswell as to assess any variability not induced by the glyphosate. Whenthe other plants reach the first trifoliate leaf stage, usually 2-3weeks after planting, glyphosate is applied at a rate equivalent of 128oz./acre (8.895 kg/ha) of Roundup®. A laboratory track sprayer has beencalibrated to deliver a rate equivalent to those conditions.

A vegetative score of 0 to 10 is used. The score is relative to theunsprayed progenies from the same R_(o) plant. A 0 is death, while a 10represents no visible difference from the unsprayed plant. A highernumber between 0 and 10 represents progressively less damage as comparedto the unsprayed plant. Plants are scored at 7, 14, and 28 days aftertreatment (DAT). The data from the analysis of one set of transformedand control soybean plants are described on Table X and show that theCP4 EPSPS gene imparts glyphosate tolerance in soybean also.

TABLE X Glyphosate tolerance in Class II EPSPS soybean transformants(P-H35S, P-FMV35S; R0 plants; Spray rate = 128 oz./acre) Vegetativescore Vector/Plant No. day 7 day 14 day 28 13640/40-11 5 6 7 13640/40-39 10 10 13640/40-7 4 7 7 control A5403 2 1 0 control A5403 1 1 0

Example 4

The CP4 EPSPS gene may be used to select transformed plant materialdirectly on media containing glyphosate. The ability to select and toidentify transformed plant material depends, in most cases, on the useof a dominant selectable marker gene to enable the preferential andcontinued growth of the transformed tissues in the presence of anormally inhibitory substance. Antibiotic resistance and herbicidetolerance genes have been used almost exclusively as such dominantselectable marker genes in the presence of the corresponding antibioticor herbicide. The nptII/kanamycin selection scheme is probably the mostfrequently used. It has been demonstrated that CP4 EPSPS is also auseful and perhaps superior selectable marker/selection scheme forproducing and identifying transformed plants.

A plant transformation vector that may be used in this scheme ispMON17227 (FIG. 16). This plasmid resembles many of the other plasmidsdescribed infra and is essentially composed of the previously describedbacterial replicon system that enables this plasmid to replicate in E.coli and to be introduced into and to replicate in Agrobacterium, thebacterial selectable marker gene (Spc/Str), and located between theT-DNA right border and left border is the CTP2-CP4 synthetic gene in theFMV35S promoter-E9 3′ cassette. This plasmid also has single sites for anumber of restriction enzymes, located within the borders and outside ofthe expression cassette. This makes it possible to easily add othergenes and genetic elements to the vector for introduction into plants.

The protocol for direct selection of transformed plants on glyphosate isoutlined for tobacco. Explants are prepared for pre-culture as in thestandard procedure as described in Example 1: surface sterilization ofleaves from 1 month old tobacco plants (15 minutes in 10%clorox+surfactant; 3×dH₂O washes); explants are cut in 0.5×0.5 cmsquares, removing leaf edges, mid-rib, tip, and petiole end for uniformtissue type; explants are placed in single layer, upside down, on MS104plates+2 ml 4COO5K media to moisten surface; pre-culture 1-2 days.Explants are inoculated using overnight culture of Agrobacteriumcontaining the plant transformation plasmid that is adjusted to a titerof 1.2×10⁹ bacteria/ml with 4COO5K media. Explants are placed into acentrifuge tube, the Agrobacterium suspension is added and the mixtureof bacteria and explants is “Vortexed” on maximum setting for 25 secondsto ensure even penetration of bacteria. The bacteria are poured off andthe explants are blotted between layers of dry sterile filter paper toremove excess bacteria. The blotted explants are placed upside down onMS104 plates+2 ml 4COO5K media+filter disc. Co-culture is 2-3 days. Theexplants are transferred to MS104+Carbenicillin 1000 mg/l+cefotaxime 100mg/l for 3 days (delayed phase). The explants are then transferred toMS104+glyphosate 0.05 mM+Carbenicillin 1000 mg/l+cefotaxime 100 mg/l forselection phase. At 4-6 weeks shoots are cut from callus and placed onMSO+Carbenicillin 500 mg/l rooting media. Roots form in 3-5 days, atwhich time leaf pieces can be taken from rooted plates to confirmglyphosate tolerance and that the material is transformed.

The presence of the CP4 EPSPS protein in these transformed tissues hasbeen confirmed by immunoblot analysis of leaf discs. The data from oneexperiment with pMON17227 is presented in the following: 139 shootsformed on glyphosate from 400 explants inoculated with AgrobacteriumABI/pMON17227; 97 of these were positive on recallusing on glyphosate.These data indicate a transformation rate of 24 per 100 explants, whichmakes this a highly efficient and time saving transformation procedurefor plants. Similar transformation frequencies have been obtained withpMON17131 and direct selection of transformants on glyphosate with theCP4 EPSPS genes has also been shown in other plant species, including,Arabidopsis, soybean, corn, wheat, potato, tomato, cotton, lettuce, andsugarbeet.

The pMON17227 plasmid contains single restriction enzyme recognitioncleavage sites (NotI, XhoI, and BstXI) between the CP4 glyphosateselection region and the left border of the vector for the cloning ofadditional genes and to facilitate the introduction of these genes intoplants.

EXAMPLE 5A

The CP4 EPSPS gene has also been introduced into Black Mexican Sweet(BMS) corn cells with expression of the protein and glyphosateresistance detected in callus.

The backbone for this plasmid was a derivative of the high copy plasmidpUC119 (Viera and Messing, 1987). The 1.3 Kb FspI-DraI pUC119 fragmentcontaining the origin of replication was fused to the 1.3 KbSmaI-HindIII filled fragment from pKC7 (Rao and Rogers, 1979) whichcontains the neomycin phosphotransferase type II gene to conferbacterial kanamycin resistance. This plasmid was used to construct amonocot expression cassette vector containing the 0.6 kb cauliflowermosaic virus (CaMV) 35S RNA promoter with a duplication of the −90 to−300 region (Kay et al., 1987), an 0.8 kb fragment containing an intronfrom a maize gene in the 5′ untranslated leader region, followed by apolylinker and the 3′ termination sequences from the nopaline synthase(NOS) gene (Fraley et al., 1983). A 1.7 Kb fragment containing the 300bp chIoroplast transit peptide from the Arabidopsis EPSP synthase fusedin a frame to the 1.4 Kb coding sequence for the bacterial CP4 EPSPsynthase was inserted into the monocot expression cassette in thepolylinker between the intron and the NOS termination sequence to formthe plasmid pMON19653 (FIG. 17).

pMON19653 DNA was introduced into Black Mexican Sweet (BMS) cells byco-bombardment with EC9, a plasmid containing a sulfonylurea-resistantform of the maize acetolactate synthase gene. 2.5 mg of each plasmid wascoated onto tungsten particles and introduced into log-phase BMS cellsusing a PDS-1000 particle gun essentially as described (Klein et al.,1989). Transformants are selected on MS medium containing 20 ppbchlorsulfuron. After initial selection on chlorsulfuron, the calli canbe assayed directly by Western blot. Glyphosate tolerance can beassessed by transferring the calli to medium containing 5mM glyphosate.As shown in Table XI, CP4 EPSPS confers glyphosate tolerance to corncallus.

TABLE XI Expression of CP4 in BMS Corn Callus-pMON 19653 CP4 expressionLine (% extract protein) 284 0.006% 287 0.036 290 0.061 295 0.073 2990.113 309 0.042 313 0.003

To measure CP4 EPSPS expression in corn callus, the following procedurewas used: BMS callus (3 g wet weight) was dried on filter paper(Whatman#1) under vacuum, reweighed, and extraction buffer (500 μl/g dryweight; 100 mM Tris, 1 mM EDTA, 10% glycerol) was added. The tissue washomogenized with a Wheaton overhead stirrer for 30 seconds at 2.8 powersetting. After centrifugation (3 minutes, Eppendorf microfuge), thesupernatant was removed and the protein was quantitated (BioRad ProteinAssay). Samples (50 μg/well) were loaded on an SDS PAGE gel (Jule,3-17%) along with CP4 EPSPS standard (10 ng), electrophoresed, andtransferred to nitrocellulose similarly to a previously described method(Padgette, 1987). The nitrocellulose blot was probed with goat anti-CP4EPSPS IgG, and developed with I-125 Protein G. The radioactive blot wasvisualized by autoradiography. Results were quantitated by densitometryon an LKB UltraScan XL laser densitomer and are tabulated below in TableX.

TABLE XII Glyphosate resistance in BMS Corn Callus using pMON 19653 #chlorosulfuron- # cross-resistant Vector Experiment resistant lines toGlyphosate 19653 253 120 81/120 = 67.5% 19653 254 80 37/80 = 46% EC9control 253/254 8  0/8 = 0%

Improvements in the expression of Class II EPSPS could also be achievedby expressing the gene using stronger plant promoters, using better 3′polyadenylation signal sequences, optimizing the sequences around theinitiation codon for ribosome loading and translation initiation, or bycombination of these or other expression or regulatory sequences orfactors.

Example 5B

The plant-expressible genes encoding the CP4 EPSPS and a glyphosateoxidoreductasease enzyme (PCT Pub. No. WO92/00377) were introduced intoembryogenic corn callus through particle bombardment. Plasmid DNA wasprepared using standard procedures (Ausubel et al., 1987),cesium-chloride purified, and re-suspended at 1 mg/ml in TE buffer. DNAwas precipitated onto M10 tungsten or 1.0 μg gold particles (BioRad)using a calcium chloride/spermidine precipitation protocol, essentiallyas described by Klein et al. (1987). The PDS1000® gunpowder gun (BioRad)was used. Callus tissue was obtained by isolating 1-2 mm long immatureembryos from the “Hi-II” genotype (Armstrong et al., 1991), or Hi-II XB73 crosses, onto a modified N6 medium (Armstrong and Green, 1985;Songstad et al., 1991). Embryogenic callus (“type-II”; Armstrong andGreen, 1985) initiated from these embryos was maintained by subculturingat two week intervals, and was bombarded when less than two months old.Each plate of callus tissue was bombarded from 1 to 3 times with eithertungsten or gold particles coated with the plasmid DNA(s) of interest.Callus was transferred to a modified N6 medium containing an appropriateselective agent (either glyphosate, or one or more of the antibioticskanamycin, G418, or paromomycin) 1-8 days following bombardment, andthen re-transferred to fresh selection media at 2-3 week intervals.Glyphosate-resistant calli first appeared approximately 6-12 weekspost-bombardment. These resistant calli were propagated on selectionmedium, and samples were taken for assays gene expression. Plantregeneration from resistant calli was accomplished essentially asdescribed by Petersen et al. (1992).

In some cases, both gene(s) were covalently linked together on the sameplasmid DNA molecule. In other instances, the genes were present onseparate plasmids, but were introduced into the same plant through aprocess termed “co-transformation”. The 1 mg/ml plasmid preparations ofinterest were mixed together in an equal ratio, by volume, and thenprecipitated onto the tungsten or gold particles. At a high frequency,as described in the literature (e.g., Schocher et al., 1986), thedifferent plasmid molecules integrate into the genome of the same plantcell. Generally the integration is into the same chromosomal location inthe plant cell, presumably due to recombination of the plasmids prior tointegration. Less frequently, the different plasmids integrate intoseparate chromosomal locations. In either case, there is integration ofboth DNA molecules into the same plant cell, and any plants producedfrom that cell.

Transgenic corn plants were produced as described above which containeda plant-expressible CP4 gene and a plant-expressible gene encoding aglyphosate oxidoreductase enzyme.

The plant-expressible CP4 gene comprised a structural DNA sequenceencoding a CTP2/CP4 EPSPS fusion protein. The CTP2/CP4 EPSPS is a genefusion composed of the N-terminal 0.23 Kb chloroplast transit peptidesequence from the Arabidopsis thaliana EPSPS gene (Klee et al. 1987,referred to herein as CTP2), and the C-terminal 1.36 Kb5-enolpyruvylshikimate-3-phosphate synthase gene (CP4) from anAgrobacterium species. Plant expression of the gene fusion produces apre-protein which is rapidly imported into chloroplasts where the CTP iscleaved and degraded (della-Cioppa et al., 1986) releasing the matureCP4 protein.

The plant-expressible gene expressing a glyphosate oxidoreductase enzymecomprised a structural DNA sequence comprising CTP1/GOXsyn gene fusioncomposed of the N-terminal 0.26 Kb chloroplast transit peptide sequencederived from the Arabidopsis thaliana SSU 1a gene (Timko et al., 1988referred to herein as CTP1), and the C-terminal 1.3 Kb synthetic genesequence encoding a glyphosate oxidoreductase enzyme (GOXsyn, asdescribed in PCT Pub. No. WO92/00377 previously incorporated byreference. The GOXsyn gene encodes the enzyme glyphosate oxidoreductasefrom an Achromobacter sp. strain LBAA which catalyzes the conversion ofglyphosate to herbicidally inactive products, aminomethylphosphonate andglyoxylate. Plant expression of the gene fusion produces a pre-proteinwhich is rapidly imported into chloroplasts where the CTP is cleaved anddegraded (della-Cioppa et al., 1986) releasing the mature GOX protein.

Both of the above described genes also include the following regulatorysequences for plant expression: (i) a promoter region comprising a 0.6Kb 35S cauliflower mosaic virus (CaMV) promoter (Odell et al., 1985)with the duplicated enhancer region (Kay et al., 1987) which alsocontains a 0.8 Kb fragment containing the first intron from the maizeheat shock protein 70 gene (Shah et al., 1985 and PCT Pub. No.WO93/19189, the disclosure of which is hereby incorporated byreference); and (ii) a 3′ non-translated region comprising a 0.3 Kbfragment of the 3′ non-translated region of the nopaline synthase gene(Fraley et al., 1983 and Depicker, et al., 1982) which functions todirect polyadenylation of the mRNA.

The above described transgenic corn plants exhibit tolerance toglyphosate herbicide in greenhouse and field trials.

Example 6

The LBAA Class II EPSPS gene has been introduced into plants and alsoimparts glyphosate tolerance. Data on tobacco transformed with pMON17206(infra) are presented in Table XIII.

TABLE XIII Tobacco Glyphosate Spray Test (pMON17206; E35S-CTP2-LBAAEPSPS; 0/4 lbs/ac) Line 7 Day Rating 33358 9 34586 9 33328 9 34606 933377 9 34611 10 34607 10 34601 9 34589 9 Samsun (Control) 4

From the foregoing, it will be recognized that this invention is onewell adapted to attain all the ends and objects hereinabove set forthtogether with advantages which are obvious and which are inherent to theinvention. It will be further understood that certain features andsubcombinations are to utility and may be employed without reference toother features and subcombinations. This is contemplated by and iswithin the scope of the claims. Since many possible embodiments may bemade of the invention without departing from the scope thereof, it is tobe understood that all matter herein set forth or shown in theaccompanying drawings is to be interpreted as illustrative and not in alimiting sense.

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1. An isolated DNA molecule which encodes an EPSPS enzyme having the sequence of SEQ ID NO:3.
 2. A The DNA molecule of claim 1 having the sequence of SEQ ID NO:2.
 3. A The DNA molecule of claim 1 having the sequence of SEQ ID NO:9.
 4. A recombinant, double-stranded DNA molecule comprising in sequence: a) a promoter which functions in plant cells to cause the production of an RNA sequence; b) a structural DNA sequence that causes the production of an RNA sequence which encodes a EPSPS enzyme having the sequence domains: -R-X₁-H-X₂-E-(SEQ ID NO:37), in which X₁ is G, S, T, C, Y, N, Q, D or E; X₂ is S or T; and -G-D-K-X₃-(SEQ ID NO:38), in which X₃ is S or T; and -S-A-Q-X₄-K-(SEQ ID NO:39), in which X₄ is A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V; and -N-X₅-T-R-(SEQ ID NO:40), in which X₅ is A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, provided that when X₁ is D, X ₂ is T, X ₃ is S, and X ₄ is V, then X ₅ is A, R, N, D, C, Q, E, G, H, I, L, K, M, F, S, T, W, Y, or V; and c) a 3′ non-translated region which functions in plant cells to cause the addition of a stretch of polyadenyl nucleotides to the 3′ end of the RNA sequence; where the promoter is heterologous with respect to the structural DNA sequence and adapted to cause sufficient expression of the encoded EPSPS enzyme to enhance the glyphosate tolerance of a plant cell transformed with the DNA molecule.
 5. A The DNA molecule of claim 4 in which the structural DNA sequence encodes a fusion polypeptide comprising an amino-terminal chloroplast transit peptide and the EPSPS enzyme.
 6. A The DNA molecule of claim 4 in which X₁ is D or N; X₂ is S or T; X₃ is S or T; X₄ is V, I or L; and X₅ is P or Q, provided that when X₁ is D, X ₂ is T, X ₃ is S, and X ₄ is V, then X ₅ is Q.
 7. A DNA molecule of claim 6 in which the structural DNA sequence encodes an EPSPS enzyme selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:41 and SEQ ID NO:43.
 8. A The DNA molecule of claim 5 in which X₁ is D or N; X₂ is S or T; X₃ is S or T; X₄ is V, I or L; and X₅ is P or Q, provided that when X₁ is D, X ₂ is T, X ₃ is S, and X ₄ is V, then X ₅ is Q.
 9. A DNA molecule of claim 8 in which the structural DNA sequence encodes an EPSPS enzyme selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:41 and SEQ ID NO:43.
 10. A The DNA molecule of claim 8 137 in which the EPSPS sequence is enzyme has the sequence set forth in SEQ ID NO:3.
 11. A The DNA molecule of claim 10 4 in which the promoter is a plant DNA virus promoter.
 12. A The DNA molecule of claim 11 in which the promoter is selected from the group consisting of CaMV35S and FMV35S promoters.
 13. A The DNA molecule of claim 10 5 in which the structural DNA sequence encodes a chloroplast transit peptide selected from the group consisting of SEQ ID NO:11 and SEQ ID NO:15.
 14. A The DNA molecule of claim 13 in which the 3′ non-translated region is selected from the group consisting of the NOS 3′ and the E9 3′ non-translated regions.
 15. A method of producing genetically transformed plants which are tolerant toward glyphosate herbicide, comprising the steps of: a) inserting into the genome of a plant cell a recombinant, double-stranded DNA molecule comprising: i) a promoter which functions in plant cells to cause the production of an RNA sequence, ii) a structural DNA sequence that causes the production of an RNA sequence which encodes an EPSPS enzyme having the sequence domains: -R-X₁-H-X₂-E-(SEQ ID NO:37), in which X₁ is G, S, T, C, Y, N, Q, D or E; X₂ is S or T; and -G-D-K-X₃-(SEQ ID NO:38), in which X₃ is S or T; and -S-A-Q-X₄-K-(SEQ ID NO:39), in which X₄ is A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V; and -N-X₅-T-R-(SEQ ID NO:40), in which X₅ is A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, provided that when X₁ is D, X ₂ is T, X ₃ is S, and X ₄ is V, then X ₅ is A, R, N, D, C, Q, E, G, H, I, L, K, M, F, S, T, W, Y or V; and iii) a 3′ non-translated DNA sequence which functions in plant cells to cause the addition of a stretch of polyadenyl nucleotides to the 3′ end of the RNA sequence; where the promoter is heterologous with respect to the structural DNA sequence and adapted to cause sufficient expression of the polypeptide to enhance the glyphosate tolerance of a plant cell transformed with the DNA molecule; b) obtaining a transformed plant cell; and c) regenerating from the transformed plant cell a genetically transformed plant which has increased tolerance to glyphosate herbicide.
 16. A The method of claim 15 in which X₁ is D or N; X₂ is S or T; X₃ is S or T; X₄ is V, I or L; and X₅ is P or Q, provided that when X₁ is D, X ₂ is T, X ₃ is S, and X ₄ is V, then X ₅ is Q.
 17. A method of claim 16 in which the structural DNA sequence encodes an EPSPS enzyme selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:41 and SEQ ID NO:43.
 18. A The method of claim 15 in which the structural DNA sequence encodes a fusion polypeptide comprising an amino-terminal chloroplast transit peptide and the EPSPS enzyme.
 19. A The method of claim 18 in which X₁ is D or N; X₂ is S or T; X₃ is S or T; X₄ is V, I or L; and X₅ is P or Q, provided that when X₁ is D, X ₂ is T, X ₃ is S, and X ₄ is V, then X ₅ is Q.
 20. A method of claim 19 in which the structural DNA sequence encodes an EPSPS enzyme selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:42 and SEQ ID NO:44.
 21. A The method of claim 19 143 in which the EPSPS enzyme is that set forth in SEQ ID NO:3.
 22. A The method of claim 21 15 in which the promoter is from a plant DNA virus.
 23. A The method of claim 22 in which the promoter is selected from the group consisting of CaMV35S and FMV35S promoters.
 24. A glyphosate-tolerant plant cell comprising a the DNA molecule of claims claim 4, 5 or 8or 10 .
 25. A The glyphosate-tolerant plant cell of claim 24 in which the promoter is a plant DNA virus promoter.
 26. A The glyphosate-tolerant plant cell of claim 25 in which the promoter is selected from the group consisting of CaMV35S and FMV35S promoters.
 27. A The glyphosate-tolerant plant cell of claim 24 selected from the group consisting of corn, wheat, rice, barley, soybean, cotton, sugarbeet, oilseed rape, canola, flax, sunflower, potato, tobacco, tomato, alfalfa, poplar, pine, eukalyptus eucalyptus, apple, lettuce, peas, lentils, grape and turf grasses.
 28. A glyphosate-tolerant plant comprising the plant cells cell of claim
 27. 29. A The glyphosate-tolerant plant of claim 28 in which the promoter is from a DNA plant virus promoter.
 30. A The glyphosate-tolerant plant of claim 29 in which the promoter is selected from the group consisting of CaMV35S and FMV35S promoters.
 31. A The glyphosate-tolerant plant of claim 30 selected from the group consisting of corn, wheat, rice, barley, soybean, cotton, sugarbeet, oilseed rape, canola, flax, sunflower, potato, tobacco, tomato, alfalfa, poplar, pine, eukalyptus eucalyptus, apple, lettuce, peas, lentils, grape and turf grasses.
 32. A method for selectively controlling weeds in a field containing a crop having plant crop seeds or plants comprising the steps of: a) planting the crop seeds or plants which are glyphosate-tolerant as a result of a recombinant double-stranded DNA molecule being inserted into the crop seed or plant, the DNA molecule having: i) a promoter which functions in plant cells to cause the production of an RNA sequence, ii) a structural DNA sequence that causes the production of an RNA sequence which encodes an EPSPS enzyme having the sequence domains: -R-X₁-H-X₂-E-(SEQ ID NO:37), in which X₁ is G, S, T, C, Y, N, Q, D or E; X₂ is S or T; and -G-D-K-X₃-(SEQ ID NO:38), in which X₃ is S or T; and -S-A-Q-X₄-K-(SEQ ID NO:39), in which X₄ is A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V; and -N-X₅-T-R-(SEQ ID NO:40), in which X₅ is A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, provided that when X₁ is D, X ₂ is T, X ₃ is S, and X ₄ is V, then X ₅ is A, R, N, D, C, Q, E, G, H, I, L, K, M, F, S, T, W, Y or V; and iii) a 3′ non-translated DNA sequence which functions in plant cells to cause the addition of a stretch of polyadenyl nucleotides to the 3′ end of the RNA sequence where the promoter is heterologous with respect to the structural DNA sequence and adapted to cause sufficient expression of the EPSPS enzyme to enhance the glyphosate tolerance of the crop plant transformed with the DNA molecule; and b) applying to the crop and weeds in the field a sufficient amount of glyphosate herbicide to control the weeds without significantly affecting the crop.
 33. A The method of claim 32 in which X₁ is D or N; X₂ is S or T; X₃ is S or T; X₄ is V, I or L; and X₅ is P or Q, provided that when X₁ is D, X ₂ is T, X ₃ is S, and X ₄ is V, then X ₅ is Q.
 34. A method of claim 33 in which the structural DNA sequence encodes an EPSPS enzyme selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:42 and SEQ ID NO:44.
 35. A The method of claim 32 in which the structural DNA sequence encodes a fusion polypeptide comprising an amino-terminal chloroplast transit peptide and the EPSPS enzyme.
 36. A The method of claim 35 in which X₁ is D or N; X₂ is S or T; X₃ is S or T; X₄ is V, I or L; and X₅ is P or Q, provided that when X₁ is D, X ₂ is T, X ₃ is S, and X ₄ is V, then X ₅ is Q.
 37. A method of claim 36 in which the structural DNA sequence encodes an EPSPS enzyme selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:41 and SEQ ID NO:43.
 38. A The method of claim 36 155 in which the DNA molecule encodes an EPSPS enzyme as set forth in SEQ ID NO:3.
 39. A The method of claim 38 32 in which the DNA molecule further comprises a promoter selected from the group consisting of the CAMV35S and FMV35S promoters.
 40. A The method of claim 39 in which the crop plant is selected from the group consisting of corn, wheat, rice, barley, soybean, cotton, sugarbeet, oilseed rape, canola, flax, sunflower, potato, tobacco, tomato, alfalfa, poplar, pine, eukalyptus eucalyptus, apple, lettuce, peas, lentils, grape and turf grasses.
 41. A The DNA molecule of claim 5 in which the structural DNA sequence encodes a chloroplast transit peptide selected from the group consisting of SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15 and SEQ ID NO:17.
 42. A The DNA molecule of claim 41 in which the chloroplast transit peptide is encoded by a DNA sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14 and SEQ ID NO:16.
 43. A The DNA molecule of claim 5 in which the structural DNA sequence encodes a chloroplast transit peptide selected from the group consisting of SEQ ID NO:11 and SEQ ID NO:15.
 44. A The DNA molecule of claim 43 in which the chloroplast transit peptide is encoded by a DNA sequence selected from the group consisting of SEQ ID NO:10 and SEQ ID NO:14.
 45. A The DNA molecule of claim 41 in which the promoter is selected from the group consisting of CaMV 35S and FMV 35S promoters.
 46. A The DNA molecule of claim 42 in which the promoter is selected from the group consisting of CaMV 35S and FMV 35S promoters.
 47. A The DNA molecule of claim 43 in which the promoter is selected from the group consisting of CaMV 35S and FMV 35S promoters.
 48. A The DNA molecule of claim 44 in which the promoter is selected from the group consisting of CaMV 35S and FMV 35S promoters.
 49. A The DNA molecule of claim 45 in which the 3′ non-translated region is selected from the group consisting of the NOS 3′ and the E9 3′ non-translated regions.
 50. A The DNA molecule of claim 46 in which the 3′ non-translated region is selected from the group consisting of the NOS 3′ and the E9 3′ non-translated regions.
 51. A The DNA molecule of claim 47 in which the 3′ non-translated region is selected from the group consisting of the NOS 3′ and the E9 3′ non-translated regions.
 52. A The DNA molecule of claim 48 in which the 3′ non-translated region is selected from the group consisting of the NOS 3′ and the E9 3′ non-translated regions.
 53. A DNA molecule of claim 49 in which the structural DNA sequence encodes an EPSPS enzyme selected from the group consisting of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:42 and SEQ ID NO:44.
 54. A DNA molecule of claim 50 in which the structural DNA sequence encodes an EPSPS enzyme selected from the group consisting of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:42 and SEQ ID NO:44.
 55. A DNA molecule of claim 51 in which the structural DNA sequence encodes an EPSPS enzyme selected from the group consisting of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:42 and SEQ ID NO:44.
 56. A DNA molecule of claim 52 in which the structural DNA sequence encodes an EPSPS enzyme selected from the group consisting of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:42 and SEQ ID NO:44.
 57. A The DNA molecule of claim 53 137 in which the structural DNA sequence contains an EPSPS encoding sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6, SEQ ID NO:41 and SEQ ID NO:43 .
 58. A The DNA molecule of claim 54 137 in which the structural DNA sequence contains an EPSPS encoding sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:41 and SEQ ID NO:43 as set forth in SEQ ID NO:9.
 59. A DNA molecule of claim 55 in which the structural DNA sequence contains an EPSPS encoding sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:41 and SEQ ID NO:43.
 60. A DNA molecule of claim 56 in which the structural DNA sequence contains an EPSPS coding sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:41 and SEQ ID NO:43.
 61. A DNA molecule of claim 49 in which the structural DNA sequence encodes an EPSPS enzyme having the sequence of SEQ ID NO:3.
 62. A DNA molecule of claim 50 in which the structural DNA sequence encodes an EPSPS enzyme having the sequence of SEQ ID NO:3.
 63. A DNA molecule of claim 51 in which the structural DNA sequence encodes an EPSPS enzyme having the sequence of SEQ ID NO:3.
 64. A DNA molecule of claim 52 in which the structural DNA sequence encodes an EPSPS enzyme having the sequence of SEQ ID NO:3.
 65. A DNA molecule of claim 61 in which the structural DNA sequence contains an EPSPS encoding sequence selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:9.
 66. A DNA molecule of claim 62 in which the structural DNA sequence contains an EPSPS encoding sequence selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:9.
 67. A DNA molecule of claim 63 in which the structural DNA sequence contains an EPSPS encoding sequence selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:9.
 68. A DNA molecule of claim 64 in which the structural DNA sequence contains an EPSPS encoding sequence selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:9.
 69. A The glyphosate-tolerant plant cell of claim 25 149 in which: (a) the promoter is selected from the group consisting of CaMV 35S and FMV 35S promoters; (b) the structural DNA sequence encodes: (i) a chloroplast transit peptide selected from the group consisting of SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15 and SEQ ID NO:17; and (ii) an EPSPS enzyme selected from the group consisting of SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7, SEQ ID NO:42 and SEQ ID NO:44 ; and (c) the 3′ non-translated region is selected from the group consisting of the NOS 3′ and the E9 3′ non-translated regions.
 70. A The glyphosate-tolerant plant cell of claim 69 in which the structural DNA sequence comprises: (a) a chloroplast transit peptide encoding DNA sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14 and SEQ ID NO:16; and (b) an EPSPS encoding sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6, SEQ ID NO:41 and SEQ ID NO:43 .
 71. A The glyphosate-tolerant plant cell of claim 69 in which the structural DNA sequence comprises: (a) a chloroplast transist peptide encoding DNA sequence selected from the group consisting of SEQ ID NO:10 and SEQ ID NO:14; and (b) a DNA sequence encoding an EPSPS enzyme having the sequence of SEQ ID NO:3.
 72. A The glyphosate-tolerant plant cell of claim 71 in which the structural DNA sequence comprises an EPSPS encoding sequence selected from the group consisting of SEQ ID NO:2 and as set forth in SEQ ID NO:9.
 73. A The glyphosate-tolerant plant cell of claim 71 selected from the group consisting of corn, wheat, rice, barley, soybean, cotton, sugarbeet, oilseed rape, canola, flax, sunflower, potato, tobacco, tomato, alfalfa, poplar, pine, eukalyptus eucalyptus, apple, lettuce, peas, lentils, grape and turf grasses.
 74. A glyphosate-tolerant plant comprising a the DNA molecule of claims 5, 8 or 10 claim 131 in which: (a) the promoter is selected from the group consisting of CaMV 35S and FMV 35S promoters; (b) the structural DNA sequence encodes: ; (i) a chloroplast transit peptide selected from the group consisting of SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15 and SEQ ID NO:17; and (ii) an EPSPS enzyme selected from the group consisting of SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7, SEQ ID NO:42 and SEQ ID NO:44 ; and (c) the 3′ non-translated region is selected from the group consisting of the NOS 3′ and the E9 3′ non-translated regions.
 75. A The glyphosate-tolerant plant of claim 74 in which the structural DNA sequence comprises: (a) a chloroplast transit peptide encoding DNA sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14 and SEQ ID NO:16; and (b) an EPSPS encoding sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6, SEQ ID NO:41 and SEQ ID NO:43 .
 76. A The glyphosate-tolerant plant of claim 75 in which the structural DNA sequence comprises: (a) a chloroplast transit peptide encoding DNA sequence selected from the group consisting of SEQ ID NO:10 and SEQ ID NO:14; and (b) a DNA sequence encoding an EPSPS enzyme having the sequence of SEQ ID NO:3.
 77. A The glyphosate-tolerant plant of claim 76 74 in which the structural DNA sequence comprises an EPSPS encoding sequence selected from the group consisting of SEQ ID NO:2 and as set forth in SEQ ID NO:9.
 78. A The glyphosate-tolerant plant of claim 77 74 selected from the group consisting of corn, wheat, rice, barley, soybean, cotton, sugarbeet, oilseed rape, canola, flax, sunflower, potato, tobacco, tomato, alfalfa, poplar, pine, eukalyptus eucalyptus, apple, lettuce, peas, lentils, grape and turf grasses.
 79. A seed of a the glyphosate-tolerant plant of claim 28, wherein the seed comprises the recombinant DNA molecule.
 80. A seed of a the glyphosate-tolerant plant of claim 31, wherein the seed comprises the recombinant DNA molecule.
 81. A seed of a the glyphosate-tolerant plant of claim 75, wherein the seed comprises the recombinant DNA molecule.
 82. A seed of a the glyphosate-tolerant plant of claim 76, wherein the seed comprises the recombinant DNA molecule.
 83. A seed of a the glyphosate-tolerant plant of claim 77, wherein the seed comprises the recombinant DNA molecule.
 84. A seed of a the glyphosate-tolerant plant of claim 78 129, wherein the seed comprises the recombinant DNA molecule.
 85. A seed of a the glyphosate-tolerant plant of claim 79 144, wherein the seed comprises the recombinant DNA molecule.
 86. A transgenic soybean plant which contains a heterologous gene which encodes an EPSPS enzyme having a K_(m) for phosphoenolpyruvate (PEP) between 1 and 150 μM and a K_(i)(glyphosate)/K_(m)(PEP) ratio between about 2 and 500, said plant exhibiting tolerance to N-phosphonomethylglycine herbicide at a rate of 1 lb/acre without significant yield reduction due to herbicide application.
 87. Seed of a soybean plant of claim
 86. 88. The DNA molecule of claim 6 in which the structural DNA sequence contains an EPSPS encoding sequence selected from the group consisting of SEQ ID NO:41 and SEQ ID NO:43.
 89. The DNA molecule of claim 8 in which the structural DNA sequence contains an EPSPS encoding sequence selected from the group consisting of SEQ ID NO:41 and SEQ ID NO:43.
 90. The method of claim 16 in which the structural DNA sequence contains an EPSPS encoding sequence selected from the group consisting of SEQ ID NO:41 and SEQ ID NO:
 43. 91. The method of claim 19 in which the structural DNA sequence encodes an EPSPS enzyme having a sequence selected from the group consisting of SEQ ID NO:42 and SEQ ID NO:44.
 92. The method of claim 33 in which the structural DNA sequence encodes an EPSPS enzyme having a sequence selected from the group consisting of SEQ ID NO:42 and SEQ ID NO:44.
 93. The method of claim 36 in which the structural DNA sequence contains an EPSPS encoding sequence selected from the group consisting of SEQ ID NO:41 and SEQ ID NO:43.
 94. The DNA molecule of claim 49 in which the structural DNA sequence encodes an EPSPS enzyme having a sequence selected from the group consisting of SEQ ID NO: 42 and SEQ ID NO:
 44. 95. The DNA molecule of claim 50 in which the structural DNA sequence encodes an EPSPS enzyme having a sequence selected from the group consisting of SEQ ID NO: 42 and SEQ ID NO:
 44. 96. The DNA molecule of claim 51 in which the structural DNA sequence encodes an EPSPS enzyme having a sequence selected from the group consisting of SEQ ID NO: 42 and SEQ ID NO:
 44. 97. The DNA molecule of claim 52 in which the structural DNA sequence encodes an EPSPS enzyme having a sequence selected from the group consisting of SEQ ID NO: 42 and SEQ ID NO:
 44. 98. The glyphosate-tolerant plant cell of claim 25 in which: a) the promoter is selected from the group consisting of CaMV 35S and FMV 35S promoters; b) the structural DNA sequence encodes: i) a chloroplast transit peptide selected from the group consisting of SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15 and SEQ ID NO:17; and ii) an EPSPS enzyme selected from the group consisting of SEQ ID NO:42 and SEQ ID NO:44; and c) the 3′ non-translated region is selected from the group consisting of the NOS 3′ and the E9 3′ non-translated regions.
 99. The glyphosate-tolerant plant cell of claim 26 in which the structural DNA sequence comprises: a) a chloroplast transit peptide encoding DNA sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14 and SEQ ID NO:16; and b) an EPSPS encoding sequence selected from the group consisting of SEQ ID NO:41 and SEQ ID NO:43.
 100. The glyphosate-tolerant plant comprising the DNA molecule of claim 4 , 5 or 8 in which: a) the promoter is selected from the group consisting of CaMV 35S and FMV 35S promoters; b) the structural DNA sequence encodes: i) a chloroplast transit peptide selected from the group consisting of SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15 and SEQ ID NO:17; and ii) an EPSPS enzyme selected from the group consisting of SEQ ID NO:42 and SEQ ID NO:44; and c) the 3′ non-translated region is selected from the group consisting of the NOS 3′ and the E9 3′ non-translated regions.
 101. The glyphosate-tolerant plant of claim 28 in which the structural DNA sequence comprises: a) a chloroplast transit peptide encoding DNA sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14 and SEQ ID NO:16; and b) an EPSPS encoding sequence selected from the group consisting of SEQ ID NO:41 and SEQ ID NO:43.
 102. An isolated DNA molecule that encodes a 5-enolpyruvylshikimate- 3 -phosphate synthase (EPSPS) enzyme having the sequence of SEQ ID NO:70.
 103. A recombinant, double-stranded DNA molecule comprising in sequence: a) a promoter which functions in plant cells to cause the production of an RNA sequence; b) a structural DNA sequence that causes the production of an RNA sequence which encodes an EPSPS enzyme having the sequence of SEQ ID NO:70; and c) a 3′ non-translated region that functions in plant cells to cause the addition of a stretch of polyadenyl nucleotides to the 3′ end of the RNA sequence; where the promoter is heterologous with respect to the structural DNA sequence and adapted to cause sufficient expression of the encoded EPSPS enzyme to enhance the glyphosate tolerance of a plant cell transformed with the DNA molecule.
 104. The DNA molecule of claim 103, wherein the structural DNA sequence further causes the production of an RNA sequence that encodes an amino-terminal chloroplast transit peptide that is fused to the EPSPS enzyme.
 105. The DNA molecule of claim 104, wherein the chloroplast transit peptide has the sequence of SEQ ID NO:11 or SEQ ID NO:15.
 106. The DNA molecule of claim 103, wherein the promoter is a plant DNA virus promoter.
 107. The DNA molecule of claim 106, wherein the promoter is a CaMV35S promoter or an FMV35S promoter.
 108. The DNA molecule of claim 103, wherein the 3′ non-translated region is a NOS 3′ or an E9 3′ non-translated region.
 109. A method of producing a genetically transformed plant which is tolerant toward glyphosate herbicide, comprising the steps of: a) inserting into the genome of a plant cell a recombinant double-stranded DNA molecule comprising: i) a promoter that functions in plant cells to cause the production of an RNS sequence; ii) a structural DNA sequence that causes the production of an RNS sequence which encodes an EPSPS enzyme having the sequence of SEQ ID NO: 70; and iii) a 3′ non-translated DNA sequence that functions in plant cells to cause the addition of a stretch of polyadenyl nucleotides to the 3′ end of the RNS sequence;  wherein the promoter is heterologous with respect to the structural DNA sequence and adapted to cause sufficient expression of the polypeptide to enhance the glyphosate tolerance of a plant cell transformed with the DNA molecule; b) obtaining a transformed plant cell; and c) regenerating from the transformed plant cell a genetically transformed plant which has increased tolerance to glyphosate herbicide.
 110. The method of claim 109, wherein the structural DNA further causes the production of an RNA sequence that encodes an amino-terminal chloroplast transit peptide that is fused to the EPSPS enzyme.
 111. The method of claim 110, wherein the chloroplast transit peptide has the sequence of SEQ ID NO:11 or SEQ ID NO:15.
 112. The method of claim 109, in which the promoter is a plant DNA virus promoter.
 113. The method of claim 112, in which the promoter is a CaMV35S promoter or an FMV35S promoter.
 114. The method of claim 109, wherein the 3′ non-translated DNA sequence is a NOS 3′ or an E9 3′ non-translated sequence.
 115. A glyphosate-tolerant plant cell comprising a DNA sequence encoding an EPSPS enzyme having the sequence of SEQ ID NO:
 70. 116. A glyphosate-tolerant plant comprising a DNA sequence encoding an EPSPS enzyme having the sequence of SEQ ID NO:
 70. 117. The plant of claim 116, wherein the plant is corn, wheat, rice, barley, soybean, cotton, sugarbeet, oilseed rape, canola, flax, sunflower, potato, tobacco, tomato, alfalfa, poplar, pine, eucalyptus, apple, lettuce, peas, lentils, grape or turf grasses.
 118. The plant of claim 117, wherein the plant is corn.
 119. The plant of claim 117, wherein the plant is soybean.
 120. The plant of claim 117, wherein the plant is canola.
 121. The plant of claim 117, wherein the plant is cotton.
 122. A seed of the plant of claim 116, wherein the seed comprises the DNA sequence encoding an EPSPS enzyme having the sequence of SEQ ID NO:
 70. 123. The seed of claim 122, wherein the seed is corn, wheat, rice, barley, soybean, cotton, sugarbeet, oilseed rape, canola, flax, sunflower, potato, tobacco, tomato, alfalfa, poplar, pine, eucalyptus, apple, lettuce, peas, lentils, grape or turf grass seed.
 124. The seed of claim 123, wherein the seed is corn seed.
 125. The seed of claim 123, wherein the seed is soybean seed.
 126. The seed of claim 123, wherein the seed is canola seed.
 127. The seed of claim 123, wherein the seed is cotton seed.
 128. A glyphosate tolerant plant cell comprising the recombinant DNA molecule of claim
 103. 129. A plant comprising the glyphosate tolerant plant cell of claim
 128. 130. A method for selectively controlling weeds in a field containing a crop having planted crop seeds or plants comprising the steps of: a) planting the crop seeds or plants which are glyphosate-tolerant as a result of a recombinant double-stranded DNA molecule being inserted into the crop seed or plant, the DNA molecule having: i) a promoter which functions in plant cells to cause the production of an RNA sequence, ii) a structural DNA sequence that causes the production of an RNA sequence which encodes an EPSPS enzyme having the sequence of SEQ ID NO:70; and iii) a 3′ non-translated DNA sequence which functions in plant cells to cause the addition of a stretch of polyadenyl nucleotides to the 3′ end of the RNA sequence,  where the promoter is heterologous with respect to the structural DNA sequence and adapted to cause sufficient expression of the EPSPS enzyme to enhance the glyphosate tolerance of the crop plant transformed with the DNA molecule; and b) applying to the crop and weeds in the field a sufficient amount of glyphosate herbicide to control the weeds without significantly affecting the crop.
 131. A recombinant, double-stranded DNA molecule comprising in sequence: a) a promoter which functions in plant cells to cause the production of an RNA sequence; b) a structural DNA sequence that causes the production of an RNA sequence which encodes an EPSPS enzyme having the sequence of SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO: 7; c) a 3′ non-translated region which functions in plant cells to cause the addition of a stretch of polyadenyl nucleotides to the 3′ end of the RNA sequence; where the promoter is heterologous with respect to the structural DNA sequence and adapted to cause sufficient expression of the encoded EPSPS enzyme to enhance the glyphosate tolerance of a plant cell transformed with the DNA molecule.
 132. The DNA molecule of claim 131 in which the structural DNA sequence encodes a fusion polypeptide comprising an amino-terminal chloroplast transit peptide and the EPSPS enzyme.
 133. The DNA molecule of claim 131 in which the promoter is a plant DNA virus promoter.
 134. The DNA molecule of claim 133 in which the promoter is selected from the group consisting of CaMV35S and FMV35S promoters.
 135. The DNA molecule of claim 132 in which the structural DNA sequence encodes a chloroplast transit peptide selected from the group consisting of SEQ ID NO: 11 and SEQ ID NO:
 15. 136. The DNA molecule of claim 131 in which the 3′ non-translated region is selected from the group consisting of the NOS 3′ and the E9 3′ non-translated regions.
 137. A method of producing genetically transformed plants which are tolerant toward glyphosate herbicide, comprising the steps of: a) inserting into the genome of a plant cell a recombinant, double-stranded DNA molecule comprising: i) a promoter which functions in plant cells to cause the production of an RNA sequence, ii) a structural DNA sequence that causes the production of an RNA sequence which encodes an EPSPS enzyme having the sequence of SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7; and iii) a 3′ non-translated DNA sequence which functions in plant cells to cause the addition of a stretch of polyadenyl nucleotides to the 3′ end of the RNA sequence;  where the promoter is heterologous with respect to the structural DNA sequence and adapted to cause sufficient expression of the polypeptide to enhance the glyphosate tolerance of a plant cell transformed with the DNA molecule; b) obtaining a transformed plant cell; and c) regenerating from the transformed plant cell a genetically transformed plant which has increased tolerance to glyphosate herbicide.
 138. The method of claim 137 in which the structural DNA sequence encodes a fusion polypeptide comprising an amino-terminal chloroplast transit peptide and the EPSPS enzyme.
 139. The method of claim 130, wherein the chloroplast transit peptide has the sequence of SEQ ID NO: 11 or SEQ ID NO:
 15. 140. The method of claim 137 in which the promoter is a plant DNA virus.
 141. The method of claim 140 in which the promoter is a CaMV35S promoter or a FMV35S promoter.
 142. The method of claim 137, wherein the 3′ non-translated DNA sequence is a NOS 3′ or an e9 3′ non-translated sequence.
 143. A glyphosate-tolerant plant cell comprising the DNA molecule of claim 131 .
 144. A plant comprising the glyphosate-tolerant plant cell of claim 143 .
 145. A glyphosate-tolerant plant cell comprising an EPSPS enzyme having the sequence of SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7.
 146. A glyphosate-tolerant plant comprising an EPSPS enzyme having the sequence of SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO:
 7. 147. The glyphosate-tolerant plant cell of claim 143 or 145 selected from the group consisting of corn, wheat, rice, barley, soybean, cotton, sugarbeet, oilseed rape, canola, flax, sunflower, potato, tobacco, tomato, alfalfa, poplar, pine, eucalyptus, apple, lettuce, peas, lentils, grape, and turf grasses.
 148. The glyphosate-tolerant plant of claim 144 or 146 selected from the group consisting of corn, wheat, rice, barley, soybean, cotton, sugarbeet, oilseed rape, canola, flax, sunflower, potato, tobacco, tomato, alfalfa, poplar, pine, eucalyptus, apple, lettuce, peas, lentils, grapes, and turf grasses.
 149. A method for selectively controlling weeds in a field containing a crop having planted crop seeds or plants comprising the steps of: a) planting the crop seeds or plants which are glyphosate-tolerant as a result of a recombinant double-stranded DNA molecule being inserted into the crop seed or plant, the DNA molecule having: i) a promoter which functions in plant cells to cause the production of an RNA sequence, ii) a structural DNA sequence that causes the production of an RNA sequence which encodes an EPSPS enzyme having the sequence of SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7; and iii) a 3′ non-translated DNA sequence which functions in plant cells to cause the addition of a stretch of polyadenyl nucleotides to the 3′ end of the RNA sequence,  wherein the promoter is heterologous with respect to the structural DNA sequence and adapted to cause sufficient expression of the EPSPS enzyme to enhance the glyphosate tolerance of the crop plant transformed with the DNA molecule; and b) applying to the crop and weeds in the field a sufficient amount of glyphosate herbicide to control the weeds without significantly affecting the crop. 